Txp40, a ubiquitous insecticidal toxin protein from Xenorhabdus and Photorhabdus bacteria.

Xenorhabdus and Photorhabdus are gram-negative bacteria that produce a range of proteins that are toxic to insects. We recently identified a novel 42-kDa protein from Xenorhabdus nematophila that was lethal to the larvae of insects such as Galleria mellonella and Helicoverpa armigera when it was injected at doses of 30 to 40 ng/g larvae. In the present work, the toxin gene txp40 was identified in another 59 strains of Xenorhabdus and Photorhabdus, indicating that it is both highly conserved and widespread among these bacteria. Recombinant toxin protein was shown to be active against a variety of insect species by direct injection into the larvae of the lepidopteran species G. mellonella, H. armigera, and Plodia interpunctella and the dipteran species Lucilia cuprina. The protein exhibited significant cytotoxicity against two dipteran cell lines and two lepidopteran cell lines but not against a mammalian cell line. Histological data from H. armigera larvae into which the toxin was injected suggested that the primary site of action of the toxin is the midgut, although some damage to the fat body was also observed.

Regulation of the rat NHE3 gene promoter by sodium butyrate.

Short-chain fatty acids, and especially butyrate (NaB), stimulate sodium and water absorption by inducing colonic Na+/H+ exchange (NHE). NaB induces NHE3 activity and protein and mRNA expression both in vivo and in vitro. NaB, as a histone deacetylase (HDAC) inhibitor, regulates gene transcription. We therefore studied whether NaB regulates transcription of the rat NHE3 promoter in transiently transfected Caco-2 cells. NaB (5 mM) strongly stimulated reporter gene activity, and this stimulation was prevented with actinomycin D, indicating transcriptional activation. NaB effects on the NHE3 promoter depended on the activity of Ser/Thr kinases, in particular, protein kinase A (PKA). However, PKA stimulation alone did not have an effect on promoter activity, and it did not act synergistically with NaB. Another HDAC inhibitor, Trichostatin A (TSA), stimulated NHE3 promoter in a Ser/Thr kinase-independent fashion. The putative NaB-responsive elements were localized within -320/-34 bp of the NHE3 promoter. These findings suggest that PKA mediates NaB effects on NHE3 gene transcription and that the mechanism of NaB action is different from that of TSA.

Molecular cloning of murine sodium-phosphate cotransporter type IIb (Na/P(i)-IIb) gene promoter and characterization of gene structure.

We report the cloning of the murine Na/P(i)-IIb cotransporter gene, which spans more than 18 kilobases and consists of 12 introns and 13 exons. Three promoter/reporter gene constructs, -159/+73, -429/+73 and -954/+73, showed significant luciferase activity (22-82-fold over background) when transfected into in rat intestinal epithelial (RIE-1) cells.

Molecular cloning of the murine PHEX gene promoter.

We report the novel cloning of the murine PHEX promoter, the gene that is mutated in X-linked hypophosphatemic rickets (XLH). Four promoter/reporter gene constructs, -133/+104, -542/+104, -1061/+104, and -2866/+104, showed significant luciferase activity (4.9-13.2-fold over background) when transfected into rat osteogenic sarcoma (UMR-106) cells.

Characterization of a juvenile hormone binding lipophorin from the blowfly Lucilia cuprina.

The larval haemolymph of the sheep blowfly Lucilia cuprina (Weidemann) contains a juvenile hormone binding protein with a Kd for racemic JH III of 33 +/- 6 nM. The density of the binding sites is 212 +/- 33 pmol/mg haemolymph protein. The binding protein is equally specific for JH III and methyl farnesoate. Some natural juvenoids were ranked for their ability to displace [3H]JH III with JH III > JH II > JH I > JH III acid > JH III diol > JHB3 = no detectable displacement. These data, together with displacement studies for 14 synthetic juvenoids, indicate some characteristics of the JH binding cleft. The binding protein is a high density lipophorin (density = 1.15 g/ml) and has subunit molecular weights of 228 kDa (apolipophorin I) and 70 kDa (apolipophorin II). The N-terminal amino acid sequences of the subunits have no discernible homology to any previously sequenced protein. Lipophorin-specific immunocytochemical staining occurs in a subset of fat body cells.

Association of exogenous DNA with cattle and insect spermatozoa in vitro.

Spermatozoa isolated from domestic cattle (Bos taurus), the Australian sheep blowfly (Lucilia cuprina), and the honeybee (Apis mellifera) are capable of binding exogenous radiolabeled linear DNA. Both motile and nonmotile bovine sperm exhibit four distinct patterns of DNA association. Following treatment with DNase I, the relative proportion of one of these patterns increases specifically in living sperm, suggesting that a small proportion of DNA that associates with bovine sperm may be sequestered within the sperm head.

The role of microtubules and cell-wall deposition in elongation of regenerating protoplasts of Mougeotia.

Protoplasts of the filamentous green alga Mougeotia sp. are spherical when isolated and revert to their normal cylindrical cell shape during regeneration of a cell wall. Sections of protoplasts show that cortical microtubules are present at all times but examination of osmotically ruptured protoplasts by negative staining shows that the microtubules are initially free and become progressively cross-bridged to the plasma membrane during the first 3 h of protoplast culture. Cell-wall microfibrils areoobserved within 60 min when protoplasts are returned to growth medium; deposition of microfibrils that is predominantly transverse to the future axis of elongation is detectable after about 6 h of culture. When regenerating protoplasts are treated with either colchicine or isopropyl-N-phenyl carbamate, drugs which interfere with microtubule polymerization, they remain spherical and develop cell walls in which the microfibrils are randomly oriented.