Drosophila P[Gal4] lines reveal that motor neurons involved in feeding persist through metamorphosis

Two P[Gal4] insertion lines in Drosophila melanogaster, MT11 and MT26, express GAL4 specifically in two to three pairs of pharyngeal motor neurons (PMN) in the suboesophageal ganglion. By using various secondary reporters, the architecture of the PMN, including their efferent axons in the pharyngeal nerve, was visualized. This allowed us to identify a pharyngeal dilator muscle as their target. To study the function of these neurons, we crossed line MT11 with a UAS-tetanus toxin gene construct (TNT-C) that inhibits all synaptic transmission. The offspring shows a reduction in food ingestion of 75% compared to the MT11 and TNT-C controls, demonstrating that PMN control food uptake. More important, lines MT11 and MT26 enabled us to follow PMN and their processes through metamorphosis, since labeling appears in the late third larval instar and persists up to adulthood. The motor axons innervate a pharyngeal muscle in the larva as well and extend through the maxillary nerve, proving that this nerve is homologous to the adult pharyngeal nerve. Efferent arborizations persist throughout metamorphosis, even though the larval muscle histolyzes by 20% of pupal life. Yet, some dedifferentiated structures remain, which may serve as a template for the formation of the adult muscle. Labeling of line MT26 with bromodeoxyuridine at embryonic or larval stages suggests that these neurons undergo their terminal mitosis in the mid to late embryo. © 1998 John Wiley & Sons, Inc. J Neurobiol 37: 237–250, 1998     

Larval chemosensory projections and invasion of adult afferents in the antennal lobe of Drosophila

We have studied the fate of olfactory afferents during metamorphic transformation of Drosophila melanogaster. Intracellular labeling of afferents from larval head chemosensilla suggests that the larval antennal lobe may be an olfactory target, whereas tritocerebral and suboesophageal centers are likely targets of gustatory sensilla. Application of monoclonal antibody 22C10 shows that the larval antennal nerve is the precursor of the adult antennal nerve and is used as a centripetal pathway for the adult afferents. Likely guidance cues are larval olfactory afferents that persist during early metamorphosis. P[GAL4] enhancer trap lines are introduced as efficient markers to follow the establishment of adult sensory projections. β-Galactosidase and the bovine TAU protein were used as reporter proteins, and their expression patterns are compared. P[GAL4] lines MT14 and KL116 demonstrate that adult antennal afferents have arrived in the antennal lobe 24 h after pupariation and extend to the contralateral lobe 6 h later. Line MT14 expresses GAL4 mostly in basiconic sensilla and in certain trichoid sensilla, whereas KL116 is specific for trichoid and a small subset of basiconic sensilla. In the antennal lobe, largely complementary subsets of glomeruli are labeled by the two lines, in agreement with the observation that particular types of sensilla project to particular target glomeruli. © 1997 John Wiley & Sons, Inc. J Neurobiol 32: 281–297, 1997.     

Cell ablation by ectopic expression of cell death genes, ced-3 and Ice, in Drosophila

We have developed a system for killing specific cells in Drosophila using ectopic expression of cell death genes. CED-3 and ICE (caspase-1) are proteins required for programmed cell death in the nematode Caenorhabditis elegans and in mammals, respectively. Our previous study has shown that both ced-3 and Ice can elicit cell death in Drosophila . By expressing ced-3 or Ice in several kinds of cells using a GAL4-UAS system and examining the resulting morphological defects, we show that these abnormalities are thought to be caused by the action of ced-3 or Ice genes. As cells are killed by apoptosis in our system, we could eliminate the possibility of harmful effects on the neighboring cells. Our system provides an alternative and novel cell ablation method to elucidate mechanisms of cell differentiation and cell-cell interactions during development in Drosophila .     

Ectopic expression of sex-peptide in a variety of tissues in Drosophila females using the P[GAL4] enhancer-trap system

Sex-peptide (SP), which is secreted by the accessory gland of Drosophila males, is transferred to the female during copulation, thereby reducing her sexual appetite (receptivity to males) and stimulating ovulation/oviposition. SP is known to be taken up into the hemolymph of mated females, but it is not clear whether there are two separate target tissues, for behavioral changes and ovulation or only one target for both responses. We have employed the GAL4-UAS system to express SP transgene constructs, both in different tissues and in different cellular components of virgin females. A cytoplasmic form of SP lacking a signal sequence did not evoke any responses, even when expressed ubiquitously. In contrast, a membrane-bound form of SP induced typical post-mating behavior, indicating that SP must be outside the cell in order to exert its biological effects. A total of 204 randomly selected P[GAL4] enhancer-trap lines were screened for their ability to induce SP responses in combination with the membrane-bound SP expressed under GAL4 control. Thirty-three lines were associated with both behavioral change and stimulated ovulation. No line was associated with only one of the two responses, implying that the SP target(s) mediating the two responses are either identical, very closely located, or present in two distinct tissues with a common set of genetic determinants. Western blot analysis of head, thorax, and abdominal extracts revealed that the biological activity was correlated with expression in the head fraction. Received: 3 October 1996 / Accepted: 6 January 1997     

Inhibition of benzo[a]pyrene-induced cytotoxicity and cytochrome P450 1A activity by dietary flavonoids in human liver cell model: structure-activity relationship

Inhibition of benzo[a]pyrene (B[a]P)-induced cytotoxicity and cytochrome p450 1A (CYP 1A) activity by flavonoids (1–100 M) was examined in terms of the structure-activity relationship in the human liver-derived cell model (HepG2). Two hydroxyl groups in the 5- and 7-position of flavonoids were essential to inhibit B[a]P-induced cytotoxicity. Generally, flavones (IC₅₀; 5.0–17.2 M) were more potent than the corresponding flavonols (IC₅₀; 42.7–131.8 M), and flavonoids such as apigenin (IC₅₀; 7.2 M) were more active than the corresponding isoflavonoids, genistein (IC₅₀; 61.7 M). The planar structure of flavone proved to be important in inhibiting B[a]P-induced toxicity and CYP 1A activity. The inhibitory effect of flavonoids on B[a]P-induced CYP 1A activity was correlated well with the inhibition of B[a]P-induced cytotoxicity (r=0.635, p<;0.01).     

Structure-activity relationships of organosulfur compounds as inhibitors of cytochrome P450 1-mediated activation of benzo[a]pyrene in a human cell model

Twelve naturally-occurring organosulfur compounds were investigated as inhibitors of cytochrome P450 1 (CYP450 1)-mediated activation of benzo[a]pyrene (B[a]P) in human hepatoma (HepG2) cells. Inhibition depended on the presence of a diallyl group and the number of S atoms. Diallyl trisulfide (DATS), with a diallyl group and three S atoms, had the highest activity with an IC₅₀ of 0.4 mM, and 1.5-fold higher potency than diallyl disulfide (DADS) containing a diallyl group and two S atoms. Organosulfur compounds containing an alkyl group were less effective, or even ineffective, inhibitors of both CYP450 1 and B[a]P-induced cytotoxicity than DADS and DATS. Alliin and S-allyl cysteine containing the S-cysteinyl group had no inhibition.     

A reversible calix[4]arene armed phenolphthalein based fluorescent probe for the detection of Zn2+ and an application in living cells

A reversible and easy assembled fluorescent sensor based on calix[4]arene and phenolphthalein (C4P) was developed for selective zinc ion (Zn²⁺) sensing in aqueous samples. The probe C4P demonstrated high selective and sensitive detection towards Zn²⁺ over other competitive metal ions. Interaction of Zn²⁺ with a solution of C4P resulted in a considerable increment in emission intensity at 440 nm (λ_ex = 365 nm) due to the suppression of photoinduced electron transfer (PET) process and the restriction of C=N isomerization . The binding constant (K_a) of C4P with Zn²⁺ was calculated to be 4.50 × 10¹¹ M^?2 and also the limit of detection of C4P for Zn²⁺ was as low as 0.108 μM (at 10^?7 M level). Moreover, the fluorescence imaging in the human colon cancer cells suggested that C4P had great potential to be used to examine Zn²⁺ in vivo.     

Benzo[a]pyrene-induced cell cycle progression is through ERKs/cyclin D1 pathway and requires the activation of JNKs and p38 mapk in human diploid lung fibroblasts

Treatment of cells with carcinogen Benzo[a]pyrene (B[a]P) allows cells to evade G1 arrest and induces cells abnormal proliferation. However, the mechanisms of its action at cellular level are not well understood. To address this question, normal human embryo lung diploid fibroblasts (HELF) were selected in the present study. We found that exposure of cells with 2.5 μM of B[a]P for 24 h resulted in a decrease of G1 population by 11.9% (P < 0.05) and a increase of S population by 17.2% (P < 0.05). Treatment of cells with B[a]P also caused dose-related activation of MAPK and induction of cyclin D1 protein expression, whereas the CDK4 protein levels were not significantly affected by B[a]P. Overexpression of cyclin D1 protein stimulated by B[a]P was significantly inhibited by 50 μM AG126 (an inhibitor of ERK1/2), but not by 25 μM SP600125 (an inhibitor of JNK1/2) or 5 μM SB203580 (an inhibitor of p38 mapk), suggesting that B[a]P-induced cyclin D1 expression was only regulated by ERK1/2 pathway. However, AG126, SP600125 or SB203580 led to cell cycle significantly arrested in G1 phase, indicating that ERK1/2, JNK1/2 and p38 mapk pathways are all required for B[a]P-induced G1/S transition. In addition, HELF cells transfecting with antisense cyclin D1 cDNA or antisense CDK4 cDNA showed significantly G1 arrest after B[a]P stimulation. These results suggested that B[a]P exposure accelerated the G1→S transition by activation of MAPK signaling pathways. Cyclin D1 and CDK4 are rate-limiting regulators of the G1→S transition and expression of cyclin D1 is predominantly regulated by ERK1/2 pathway in HELF cells.     

Longitudinal analysis of VP7 gene of group A human rotavirus G2P[4] strains circulating in the pre‐vaccine era in Sapporo,Japan from 1991 to 2011

Sequence analysis of the VP7 gene in 23 group A human rotavirus G2P[4] strains obtained during 1991–2011, that is, the pre‐vaccine era, in Sapporo, Japan showed considerable genetic diversity, mainly in variable regions. Recent G2P[4] epidemic strains were located in sublineage IVa with a distinctive substitution of D96N. This study provides background data on the genetic variability of G2P[4] rotavirus‐VP7 gene prior to the widespread use of rotavirus vaccines in Japan.     

Chemical rescue of a site-modified ligand to a [4Fe-4S] cluster in PsaC, a bacterial-like dicluster ferredoxin bound to Photosystem I

Chemical rescue of site-modified amino acids using externally supplied organic molecules represents a powerful method to investigate structure-function relationships in proteins. Here we provide definitive evidence that aryl and alkyl thiolates, reagents typically used for in vitro iron-sulfur cluster reconstitutions, serve as rescue ligands to a site-specifically modified [4Fe-4S]^1+,2+ cluster in PsaC, a bacterial dicluster ferredoxin-like subunit of Photosystem I. PsaC binds two low-potential [4Fe-4S]^1+,2+ clusters termed F_A and F_B. In the C13G/C33S variant of PsaC, glycine has replaced cysteine at position 13 creating a protein that is missing one of the ligating amino acids to iron-sulfur cluster F_B. Using a variety of analytical techniques, including non-heme iron and acid-labile sulfur assays, and EPR, resonance Raman, and Mössbauer spectroscopies, we showed that the C13G/C33S variant of PsaC binds two [4Fe-4S]^1+,2+ clusters, despite the absence of one of the biological ligands. ¹⁹F NMR spectroscopy indicated that the external thiolate replaces cysteine 13 as a substitute ligand to the F_B cluster. The finding that site-modified [4Fe-4S]^1+,2+ clusters can be chemically rescued with external thiolates opens new opportunities for modulating their properties in proteins. In particular, it provides a mechanism to attach an additional electron transfer cofactor to the protein via a bound, external ligand.     

Synthesis and characterization of de novo designed peptides modelling the binding sites of [4Fe-4S] clusters in photosystem I

Photosystem I (PS I) converts the energy of light into chemical energy via transmembrane charge separation. The terminal electron transfer cofactors in PS I are three low-potential [4Fe-4S] clusters named F_X, F_A and F_B, the last two are bound by the PsaC subunit. We have modelled the F_A and F_B binding sites by preparing two apo-peptides (maquettes), sixteen amino acids each. These model peptides incorporate the consensus [4Fe-4S] binding motif along with amino acids from the immediate environment of the iron-sulfur clusters F_A and F_B. The [4Fe-4S] clusters were successfully incorporated into these model peptides, as shown by optical absorbance, EPR and Mössbauer spectroscopies. The oxidation-reduction potential of the iron-sulfur cluster in the F_A-maquette is − 0.44 ± 0.03 V and in the F_B-maquette is − 0.47 ± 0.03 V. Both are close to that of F_A and F_B in PS I and are considerably more negative than that observed for other [4Fe-4S] model systems described earlier (Gibney, B. R., Mulholland, S. E., Rabanal, F., and Dutton, P. L. Proc. Natl. Acad. Sci. U.S.A. 93 (1996) 15041-15046). Our optical data show that both maquettes can irreversibly bind to PS I complexes, where PsaC-bound F_A and F_B were removed, and possibly participate in the light-induced electron transfer reaction in PS I.