Voltage-dependent calcium channels in the corpora allata of the adult male loreyi leafworm, Mythimna loreyi

In the corpora allata (CA) of the adult male loreyi leafworm, Mythimna loreyi, juvenile hormone acid (JHA) biosynthesis and release show a dose dependence on extracellular Ca(2+) concentration. Maxima are obtained with Ca(2+) concentrations of 2-10 mM, and synthesis and release are significantly inhibited under a Ca(2+)-free condition. The Ca(2+)-free inhibition of JHA release can be reversed by returning the glands to medium at 5 mM Ca(2+). The cytosolic free Ca(2+) concentration ([Ca(2+)](i)), which was measured with fura-2, in individual CA cells also shows a dose dependence on extracellular Ca(2+) concentration, with significant [Ca(2+)](i) depression being observed in the absence of extracellular Ca(2+).High K(+) significantly increases the JHA release and causes a transient [Ca(2+)](i) increase within seconds in CA cells. High-K(+)-stimulated JHA release is partially inhibited by the benzothiazepine (BTZ)-, dihydropyridine (DHP)- and phenylalkylamine (PAA)-sensitive L-type voltage-dependent calcium channel (VDCC) antagonists diltiazem, nifedipine and verapamil, respectively; by the N- and P/Q-type VDCC antagonist omega-conotoxin (omega-CgTx) MVIIC; and by the T-type VDCC antagonist amiloride. The N-type antagonist omega-CgTx GVIA is the most potent in inhibiting the high-K(+)-stimulated JHA release. No inhibitory effect is shown by the P-type antagonist omega-agatoxin TK (omega-Aga TK). The high-K(+)-induced transient [Ca(2+)](i) increase is largely inhibited by the L-type antagonists (diltiazem, nifedipine, verapamil), by the N- and P/Q-type antagonist omega-CgTx MVIIC and by the T-type antagonist amiloride, and is totally inhibited by the N-type antagonist omega-CgTx GVIA. No inhibitory effect is shown by the P-type antagonist omega-Aga TK.We hypothesize that L-type, N-type and T-type VDCCs may be involved to different degrees in the high-K(+)-stimulated JHA release and transient [Ca(2+)](i) increase in the individual CA cells of the adult male M. loreyi, and that the N-type VDCCs may play important roles in these cellular events.     

Involvement of tachykinin NK(1) and NK(2) receptors in changes in lung mechanics and airway microvascular leakage during the early phase of endotoxemia in Guinea pigs

We investigated the role of tachykinins in airway neurogenic responses occurring in the early phase of endotoxemia. Forty-eight anesthetized guinea pigs were evenly divided into six groups pretreated with either saline vehicle, CP-96,345 (a tachykinin NK(1) receptor antagonist), SR-48,968 (a tachykinin NK(2) receptor antagonist) or CP-96,345 and SR-48,968 in combination. Animals then received an intravenous injection of either saline (the vehicle for endotoxin) or endotoxin (30 mg/kg). Total lung resistance (R(L)) and dynamic lung compliance (C(dyn)) were continuously measured before and 30 min after administration of saline or endotoxin. Airway microvascular leakage was assessed at the end of the observation period. Endotoxin significantly increased R(L) and decreased C(dyn) 10 min after intravenous endotoxin injection. Plasma extravasation significantly increased in the trachea, main bronchi and intrapulmonary airways with endotoxin administration. These changes in lung mechanics were abolished by SR-48,968, but were unaffected by CP-96,345. The plasma extravasation was largely attenuated by CP-96,345 and/or SR-48,968. We conclude that (1) endogenous tachykinins play an important role in producing changes in lung mechanics and airway microvascular leakage during the early phase of endotoxemia and (2) activation of tachykinin NK(2) receptors is responsible for the former response, while activation of both tachykinin NK(1) and NK(2) receptors is involved in the latter response.     

Recent progress and problems in animal cloning

It is remarkable that mammalian somatic cell nuclei can form whole individuals if they are transferred to enucleated oocytes. Advancements in nuclear transfer technology can now be applied for genetic improvement and increase of farm animals, rescue of endangered species, and assisted reproduction and tissue engineering in humans. Since July 1998, more than 200 calves have been produced by nuclear transfer of somatic cell nuclei in Japan, but half of them were stillborn or died within several months of parturition. Morphologic abnormalities have also been observed in cloned calves and embryonic stem cell-derived mice. In this review, we discuss the present situation and problems with animal cloning and the possibility for its application to human medicine.     

Crystallographic studies on damaged DNAs IV. N( 4)-methoxycytosine shows a second face for Watson-Crick base-pairing, leading to purine transition mutagenesis

To investigate the mutation mechanism of purine transitions in DNA damaged with methoxyamine, a DNA dodecamer with the sequence d(CGCAAATTmo⁴CGCG), where mo⁴C is 2′-deoxy-N⁴-methoxycytidine, has been synthesized and the crystal structure determined by X-ray analysis. The duplex structure is similar to that of the original undamaged B-form dodecamer, indicating that the methoxylation does not affect the overall DNA conformation. Electron density maps clearly show that the two mo⁴C residues form Watson–Crick-type base pairs with the adenine residues of the opposite strand and that the methoxy groups of mo⁴C adopt the anti conformation to N³ around the C⁴–N⁴ bond. For the pair formation through hydrogen bonds the mo⁴C residues are in the imino tautomeric state. Together with previous work, the present work establishes that the methoxylated cytosine residue can present two alternate faces for Watson–Crick base-pairing, thanks to the amino↔imino tautomerism allowed by methoxylation. Based on this property, two gene transition routes are proposed.     

Developmental potential of cumulus cell-derived culture frozen in a quiescent state after nucleus transfer

An efficient method for freezing donor cells is necessary when using nucleus transfer of somatic cells for large-scale cloning. In the present study, we developed a method for freezing and thawing bovine cumulus cell-derived cultured cells to be used as nucleus donors. Cumulus cells were obtained from ovaries of living and slaughtered bovine and cultured in vitro. Cumulus cell-derived cultured cells were serum-starved for several days to induce a quiescent state and then frozen at -70 degrees C for at least 2 d. Immediately thereafter or 2 h after thawing, the cells were used as donor cells for nuclear transfer without additional in vitro culture. The fusion rate with recipient cytoplasts was not affected by the cumulus cell source (slaughtered or living) or time after thawing (0 and 2 h). The cleavage rate of frozen-thawed cumulus cell-derived cultured cells from slaughtered cows immediately after thawing (0 h) was highest (97%) and was significantly higher than that of controls (85%) or cells transferred 2 h after thawing (85%). There were no significant differences among any of the groups in the potential of the nuclear transfer embryos to develop into blastocysts (34 vs 44 and 44%, 39 vs 45 and 46%). Thus, storage of bovine cumulus cell-derived cultured cells in the quiescent state at -70 degrees C is effective and might be useful and convenient for large-scale cloning. The maximum storage periods and developmental potential of embryos after such nucleus transfers requires further examination.     

Allatotropic activity in the suboesophageal ganglia and corpora cardiaca of the adult male loreyi leafworm, Mythimna loreyi

Allatotropic activity was found in the methanolic extract of the suboesophageal ganglia (SOG) and the corpora cardiaca (CC) of the Mythimna loreyi virgin males. No allatotropic activity was observed in the extract of brain or corpora allata (CA). Although CA can be activated by the SOG and CC extract, respectively, CC extract inhibited the response to the SOG extract. A significant in vitro allatotropic effect was exerted by the SOG and CC extract within 10 and 15 min, respectively, and this effect can be sustained for several hours even after transferring to fresh medium without extracts. The time course pattern of the CA activation ratio in both the SOG and CC extract-treated group is very similar to, but with significantly higher level than, that in the control group, suggesting the existence of an intrinsic pacemaker or an in vitro effect that controls the fluctuation of the CA biosynthetic activity. Synthetic Manduca sexta allatotropin had no significant effect on the M. loreyi CA. The results of treatment with the adenylate cyclase activator forskolin, the phosphodiesterase inhibitor IBMX, and the cAMP analogue dibutyryl-cAMP did not indicate that cAMP might be involved in the allatotropic control of CA. Arch.     

Observations of rotation within the F(o)F(1)-ATP synthase: deciding between rotation of the F(o)c subunit ring and artifact

Annexin-I (ANX-I) is a 37-kDa protein with a calcium-dependent phospholipid-binding property. Previously we have observed the inhibition of cytosolic phospholipase A2 (cPLA2) by ANX-I in the studies using purified recombinant ANX-I, and proposed a specific interaction model for the mechanism of cPLA2 inhibition by ANX-I [Kim et al. (1994) FEBS Lett. 343, 251-255]. Here we have studied the role of ANX-I in the cPLA2 signaling pathway by transient transfection assay. The stimulation of Rat2 fibroblast cells with phorbol 12-myristate 13-acetate (PMA) induced the c-fos serum response element (SRE). The SRE stimulation by PMA was dramatically reduced by (1) pretreatment with a cPLA2-specific inhibitor, arachidonyltrifluoromethyl ketone, or (2) co-transfection with antisense cPLA2 oligonucleotide, indicating that the SRE activation was through cPLA2 activation. Co-transfection with an ANX-I expression vector also reduced the SRE stimulation by PMA, suggesting the inhibition of cPLA2 by ANX-I. The active domain of ANX-I was mapped using various deletion mutants. ANX-I(1-113) and ANX-I(34-346) were fully active, whereas ANX-I(114-346) abolished the activity. Therefore the activity was in the amino acid 34 to 113 region, which corresponds to the conserved domain I of ANX-I.     

Wood smoke-induced airway hyperreactivity in guinea pigs: time course, and role of leukotrienes and hydroxyl radical

A prior airway exposure to wood smoke induces a tachykinin-dependent increase in airway responsiveness to the subsequent smoke inhalation in guinea pigs (Life Sci. 63: 1513, 1998). To further investigate the time course of, and the contribution of other chemical mediators to, this smoke-induced airway hyperresponsiveness (SIAHR), two smoke challenges (each 10 ml) separated by 30 min were delivered into the lungs of anesthetized guinea pigs by a respirator. In the control animals, the SIAHR was evidenced by the bronchoconstrictive response to the second smoke challenge (SM2) which was approximately 5.2-fold greater than that to the first challenge (SM1). This SIAHR was alleviated by shortening the elapsed time between SM1 and SM2 to 10 min or by extending it to 60 min, and was abolished by extending it to 120 min. This SIAHR was reduced by pretreatment with either MK-571 (a leukotriene D4-receptor antagonist) or dimethylthiourea (a hydroxyl radical scavenger), but was not affected by pretreatment with either pyrilamine (a histamine H1-receptor antagonist) or indomethacin (a cyclooxygenase inhibitor). The smoke-induced reduction in the neutral endopeptidase activity (a major enzyme for tachykinin degradation) measured in airway tissues excised 30 min post SM1 was largely prevented by pretreatment with dimethylthiourea. However, this reduction was not seen in airway tissues excised 120 min post SM1. These results suggest that 1) the SIAHR to inhaled wood smoke has a rapid onset time following smoke inhalation and lasts for less than two hours, 2) leukotrienes and hydroxyl radical may play contributory roles in the development of this SIAHR, and 3) hydroxyl radical is the major factor responsible for the smoke-induced inactivation of airway neutral endopeptidase, which may possibly participate in the development of this SIAHR.     

Vegetation restoration by seasonal exclosure in the Kerqin Sandy Land,Inner Mongolia

Grazing control has been reported to be effective for the control of desertification in semi-arid regions. However, economic reasons often make complete inhibition of grazing (complete exclosure) difficult to carry out. Grazing control has been applied to the Kerqin Sandy Lands, Inner Mongolia, China, by means of seasonal exclosure, whereby grazing is allowed from November to April. The harvesting of hay is also allowed once during September - October. The aim of the reported study was to evaluate the effectiveness of this seasonal exclosure on vegetation restoration. Species compositional data were obtained from 356 quadrats and ordinated by Detrended Correspondence Analysis (DCA). Ordination indicated that landform was the most important factor influencing the species composition of the vegetation. Regardless of landform and type of grazing control, however, vegetation coverage, vegetation height and species richness were higher at sites where grazing had been controlled, than at sites lacking any control. Perennial species were dominant at the former while annual species were dominant at the latter. Both shrub and tree species were quite rare at the sites where seasonal exclosure had been carried out. It is concluded that seasonal exclosure is sufficient to restore and maintain grassland vegetation in and around the study area. When shrubby or tree vegetation is needed for reasons such as fixing sands or preventing sand dune remobilization, complete exclosure is recommended.