Expression of a catalytic domain of a Neocallimastix frontalis endoxylanase gene (xyn3) in Kluyveromyces lactis and Penicillium roqueforti

A cDNA fragment encoding the A catalytic domain of the Neocallimastix frontalis endoxylanase XYN3 was amplified and cloned by the polymerase chain reaction technique. The xyn3A DNA fragment was inserted between the Saccharomyces cerevisiae phosphoglycerate kinase gene promoter and terminator sequences on a multicopy episomal plasmid for Kluyveromyces lactis. The XYN3A domain was successfully expressed in K. lactis and functional endoxylanase was secreted by the yeast cells with the K. lactis killer toxin secretion signal. The XYN3A domain was also expressed in a strain of Penicillium roqueforti as a fusion protein (ShBLE::XYN3A) of the phleomycin-resistance gene product and the endoxylanase. Active endoxylanase was efficiently secreted from the fungal cells with the Trichoderma viride cellobiohydrolase (CBH1) secretion signal and processed by a related KEX2 endoprotease of the secretion pathway. Several differently glycosylated forms of the recombinant enzymes were secreted by the yeast and the filamentous fungus. Received: 10 November 1998 / Received revision: 8 March 1999 / Accepted: 14 March 1999     

Outcrossing as an explanation of the apparent unconventional genetic behavior of Arabidopsis thaliana hth mutants

The reappearance of HTH alleles in the offspring of homozygous Arabidopsis hth mutants is not consistent with classical Mendelian genetics. It has been suggested that stored RNA may be used to restore genetic information. However, Peng et al. reported that hth mutants tend to display outcrossing and suggested that outcrossing might provide an alternative explanation for the apparent genetic instability. We have confirmed and extended these results, corroborating that the apparent non-Mendelian behavior of hth mutants can be explained by their susceptibility to outcrossing.     

Conformational change induced by ATP binding in the multidrug ATP-binding cassette transporter BmrA

ATP-binding cassette (ABC) transporters are involved in the transport of a wide variety of substrates, and ATP-driven dimerization of their nucleotide binding domains (NBDs) has been suggested to be one of the most energetic steps of their catalytic cycle. Taking advantage of the propensity of BmrA, a bacterial multidrug resistance ABC transporter, to form stable, highly ordered ring-shaped structures [Chami et al. (2002) J. Mol. Biol. 315, 1075-1085], we show here that addition of ATP in the presence of Mg2+ prevented ring formation or destroyed the previously formed rings. To pinpoint the catalytic step responsible for such an effect, two classes of hydrolysis-deficient mutants were further studied. In contrast to hydrolytically inactive glutamate mutants that behaved essentially as the wild-type, lysine Walker A mutants formed ring-shaped structures even in the presence of ATP-Mg. Although the latter mutants still bound ATP-Mg, and even slowly hydrolyzed it for the K380R mutant, they were most likely unable to undergo a proper NBD dimerization upon ATP-Mg addition. The ATP-driven dimerization step, which was still permitted in glutamate mutants and led to a stable conformation suitable to monitor the growth of 2D crystals, appeared therefore responsible for destabilization of the BmrA ring structures. Our results provide direct visual evidence that the ATP-induced NBD dimerization triggers a conformational change large enough in BmrA to destabilize the rings, which is consistent with the assumption that this step might constitute the "power stroke" for ABC transporters.     

Phase resetting analysis of high potassium epileptiform activity in CA3 region of the rat hippocampus

The theory of phase resetting can reveal important information about the dynamic behavior of a periodic system when a single brief stimulus is applied to that system at the appropriate time. Phase resetting studies have revealed the existence in some biological systems of a vulnerable stimulus window generating desynchronization and suppression of the activity. The objective of this study was to test the hypothesis that a "singular" stimulus could annihilate this activity. Perfusion with the high-K solution produced synchronous, quasi-periodic population bursts with inter-burst interval of ~0.8-1.5 seconds. A single 0.1 ms duration anodic pulse of programmable delay and magnitude was applied to the somatic layer of the CA3 pyramidal cells. Three types of phase-resetting behavior were observed: (1) Weak resetting with little or no effect on the timing of the subsequent burst, (2) Strong resetting where the applied current pulse delayed the next event by one time period, (3) Singular behavior where the applied pulse partially or completely suppressed the subsequent bursting. The singular stimulus parameter window, however, was very narrow making it difficult to generate the singular behavior reliably. Nevertheless, the results indicate that singularities exist for high potassium neural activity and that a well timed pulse applied with the right amplitude can suppress this activity. This study suggests that phase resetting of a population of neurons is possible for quasi-periodic interictal activity and similar techniques could be applied to the control of epileptic seizures.     

Zone-specific cell proliferation during compensatory adrenal growth in rats

Compensatory adrenal growth after unilateral adrenalectomy (ULA) leads to adrenocortical hyperplasia. Because zonal growth contributions are not clear, we characterized the phenotype of cortical cells that proliferate using immunofluorescence histochemistry and zone-specific cell counting. Rats underwent ULA, sham adrenalectomy (sham), or no surgery and were killed at 2 or 5 days. Adrenals were weighed and sections immunostained for Ki67 (proliferation), cytochrome P-450 aldosterone synthase (P450aldo, glomerulosa), and cytochrome P-450 11beta-hydroxylase (P45011beta, fasciculata). Unbiased stereology was used to count proliferating glomerulosa and fasciculata cells. Adrenal weight increased after ULA compared with sham and no surgery at both time points, and there was no difference between sham and no surgery. However, either ULA or sham increased Ki67-positive cells in the outer fasciculata at both time points compared with no surgery. Outer fasciculata-restricted proliferation is thus associated with adrenal weight gain in ULA but not sham. Experiment repetition using proliferating cell nuclear antigen and bromodeoxyuridine showed similar results. After ULA, adrenal DNA, RNA, and protein increased at both time points, whereas after sham, only adrenal DNA increased at 2 days. Compensatory growth thus results from hyperplasia and hypertrophy, whereas sham induces only a transient adrenal hyperplasia. Dexamethasone pretreatment prevented the increase in adrenal weight after ULA and blocked Ki67 labeling in the outer fasciculata but not zona glomerulosa in all groups. These results clearly show that the outer fasciculata is the primary adrenal zone responsible for compensatory growth, responding to steroid-suppressible stress signals that alone are ineffective in increasing adrenal mass.     

Interhelical spacing in liquid crystalline spermine and spermidine-DNA precipitates

The structure of polyamines-DNA precipitates was studied by x-ray diffraction. Precise measurements of the interhelix distance a(H) were obtained at different NaCl, polyamine, and DNA concentrations. Most of the results were obtained using spermine and few others using spermidine. The precipitates are liquid crystalline, either hexagonal and/or cholesteric, with an interhelical spacing that depends on the ionic concentrations and on the polyamine type. In our experimental conditions, the spacing varies from 28.15 to 33.4 angstroms. This variation is interpreted in terms of different ionic components that are present inside the precipitates and that are thought to regulate the value of the cohesive energy of DNA. These results are discussed in relation to the biological processes requiring a closeness of double helices and to the role played by polyamine analogs in cancer therapy.     

Granzyme B binds to target cells mostly by charge and must be added at the same time as perforin to trigger apoptosis

Perforin (PFN) delivery of granzymes (Gzm) into the target cell at the immunological synapse is the major pathway for inducing apoptosis of virus-infected cells and tumors. A validated model for how PFN delivers Gzm into the cytosol is still lacking. PFN was originally thought to work by forming pores in the target cell plasma membrane that allow Gzm entry. This model was questioned when it was shown that GzmB is endocytosed without PFN. Moreover, apoptosis could be triggered by adding PFN to washed cells that have previously endocytosed GzmB. In this study, we show that GzmB binds to the plasma membrane mostly via nonspecific charge interactions. Washing in saline does not remove bound Gzm. However, if externally bound GzmB is completely removed, subsequent addition of PFN does not release previously endocytosed GzmB and does not trigger apoptosis. Therefore, PFN must be coendocytosed with GzmB to deliver it into the cytosol.