Preliminary observations on change of sex by the coral-inhabiting snails Coralliophila violacea (Lamarck) (Gastropoda: Coralliophilidae)

The coral-inhabiting snails, Coralliophila violacea (Lamarck), are widely distributed in the Indo–Pacific region. It has been inferred from field data that sex changes have occurred and might be influenced by neighbors. In this study, we designed a field experiment with 2 treatments, i.e. ‘single males' and ‘male–female pairings' to test the hypothesis of sex changes as well as the role of neighbors on sex changes. Snails were collected by SCUBA diving from the surface of massive coral Porites lobata in shallow waters (<6 m) in southern Taiwan. A total of 73 males and 32 females were marked and returned to the surface of a colony of the massive coral, P. lobata. After 4–5 months, a total of 25 marked males and eight marked females were recaptured. In the treatment of ‘single males', eight of 17 males had changed their sex. None of eight recaptured males in the treatment of ‘male–female pairings' had changed their sex. The occurrence of sex changes is dependent on the presence or absence of a female neighbor (P<0.05, Fisher's exact test). In this study, we have provided direct evidence of socially controlled sex changes in Coralliophila violacea.     

Spin trapping in biological systems. Oxidation of the spin trap 5,5-dimethyl-1-pyrroline-1-oxide by a hydroperoxide-hematin-system.

We have used the spin trap 5,5-dimethyl-1-pyrroline-1-oxide to determine if primary free radicals are involved in the hematin-cumene hydroperoxide system which has been shown to oxidize N-hydroxy-2-acetylaminofluorene into the nitroxyl free radical form of this carcinogen. We have found that the spin trap was oxidized itself rather than trapping either primary free radicals or carcinogen free radicals.     

Separate locations of urocortin and its receptors in mouse testis: function in male reproduction and the relevant mechanisms.

Urocortin (UCN), a newly identified corticotrophin-releasing-factor (CRF) related peptide, has been demonstrated to play important roles in female reproductive system. However, few studies were reported about its effects on male reproduction. This study aimed to investigate the expression profile of UCN and CRF receptors (CRFR) in mouse testis and functions of UCN in male reproduction. Expression of UCN and CRFR mRNA was detected by RT-PCR. Localization of UCN peptide was determined by immunohistochemistry and double-immunostaining. We found that both UCN mRNA and peptide were obviously expressed in mature spermatozoa, whereas CRFR1 and CRFR2 were expressed respectively in spermatocytes and spermatogonia. Double-immunostaining results showed that UCN expression decreased with acrosome reaction (AR) proceeding. UCN significantly inhibited AR initiated by progesterone with chlortetracycline staining and decreased spermatozoa motility concentration-dependently. Pre-incubation of spermatozoa with astressin, a CRFR antagonist, did not affect these inhibitions. In addition, flow cytometry showed that UCN concentration-dependently decreased intracellular Ca(2+) [Ca(2+)](i) in spermatozoa. In summary, UCN located in mouse spermatozoa and exerted inhibitory effects on male reproductive functions including motility and AR. UCN's inhibition on [Ca(2+)](i) via T-type calcium channels might be responsible for these effects.     

3-Carbamoyl-α-picolinic acid production by imidase-catalyzed regioselective hydrolysis of 2,3-pyridinedicarboximide in a water-organic solvent, two-phase system

3-Carbamoyl-α-picolinic acid, a versatile building block for the synthesis of agrochemicals and pharmaceuticals, was prepared by imidase-catalyzed regiospecific hydrolysis of 2,3-pyridinedicarboximide with intact Arthrobacter ureafaciens O-86 cells. Reactions were carried out in a water-organic solvent, two-phase system containing cyclohexanone at low pH to avoid spontaneous random hydrolysis. Under the optimized conditions, with the periodic addition of 2,3-pyridinedicarboximide (in total, 40 mM), the 3-carbamoyl-α-picolinic acid yield reached 36.6 mM in the water phase, with a molar conversion yield of 91.5% and a regioisomeric purity of 94.5%, in 2 h at pH 5.5. Received: 7 February 2000 / Received revision: 13 March 2000 / Accepted: 3 April 2000     

Breeding of retroviruses by DNA shuffling for improved stability and processing yields

Manufacturing of retroviral vectors for gene therapy is complicated by the sensitivity of these viruses to stress forces during purification and concentration. To isolate viruses that are resistant to these manufacturing processes, we performed breeding of six ecotropic murine leukemia virus (MLV) strains by DNA shuffling. The envelope regions were shuffled to generate a recombinant library of 5 x 106 replication-competent retroviruses. This library was subjected to the concentration process three consecutive times, with amplification of the surviving viruses after each cycle. Several viral clones with greatly improved stabilities were isolated, with the best clone exhibiting no loss in titer under conditions that reduced the titers of the parental viruses by 30- to 100-fold. The envelopes of these resistant viruses differed in DNA and protein sequence, and all were complex chimeras derived from multiple parents. These studies demonstrate the utility of DNA shuffling in breeding viral strains with improved characteristics for gene therapy.     

Leishmania amazonensis-dendritic cell interactions in vitro and the priming of parasite-specific CD4(+) T cells in vivo

The progressive disease following Leishmania amazonensis infection in mice requires functional CD4(+) T cells, which are primed to a disease-promoting phenotype during the infection. To understand how these pathogenic T cells are generated and the role of dendritic cells (DCs) in this process, we use DCs of susceptible BALB/c and resistant C3H/HeJ mice to examine parasite-DC interactions in vitro as well as the effector phenotype of T cells primed by parasite-exposed DCs in vivo. Our results demonstrate that amastigotes and metacyclics efficiently enter and activate DCs of both genetic backgrounds. Infection with amastigotes fails to induce CD40-dependent IL-12 production, but rather potentiates IL-4 production in BALB/c DCs. Upon transfer into syngeneic recipients, amastigote-exposed BALB/c DCs prime parasite-specific Th cells to produce significantly higher levels of IL-4 and IL-10 than their C3H/HeJ counterparts. Transfer studies with IL-4(-/-) DCs indicate that this enhanced Th2 priming seen in BALB/c mice is partially due to the IL-4 production by amastigote-carrying DCs. These results suggest that L. amazonensis amastigotes may condition DCs of a susceptible host to a state that favors activation of pathogenic CD4(+) T cells, and thereby provide a new perspective on the pathogenesis of cutaneous leishmaniasis and protozoan parasite-host interactions in general.     

Activation of NF-kappaB in airway epithelial cells is dependent on CFTR trafficking and Cl- channel function

Polymorphonuclear leukocyte-dominated airway inflammation is a major component of cystic fibrosis (CF) lung disease and may be associated with CF transmembrane conductance regulator (CFTR) dysfunction as well as infection. Mutant DeltaF508 CFTR is mistrafficked, accumulates in the endoplasmic reticulum (ER), and may cause "cell stress" and activation of nuclear factor (NF)-kappaB. G551D mutants also lack Cl- channel function, but CFTR is trafficked normally. We compared the effects of CFTR mutations on the endogenous activation of an NF-kappaB reporter construct. In transfected Chinese hamster ovary cells, the mistrafficked DeltaF508 allele caused a sevenfold activation of NF-kappaB compared with wild-type CFTR or the G551D mutant (P < 0.001). NF-kappaB was also activated in 9/HTEo-/pCep-R cells and in 16HBE/pcftr antisense cell lines, which lack CFTR Cl- channel function but do not accumulate mutant protein in the ER. This endogenous activation of NF-kappaB was associated with elevated interleukin-8 expression. Impaired CFTR Cl- channel activity as well as cell stress due to accumulation of mistrafficked CFTR in the ER contributes to the endogenous activation of NF-kappaB in cells with the CFTR mutation.