Southern analysis of BT-R 1, the Manduca sexta gene encoding the receptor for the Cry1Ab toxin of Bacillus thuringiensis

Various subspecies of the gram-positive bacterium Bacillus thuringiensis are known to produce a wide array of insecticidal crystal proteins (ICPs) upon sporulation. These ICPs act primarily on the brush border of midgut epithelial cells of susceptible larvae. Recently, a protein of 210?kDa, isolated from the midgut of Manduca sexta, has been demonstrated to bind the Cry1Ab toxin produced by B. thuringiensis subsp. berliner and is therefore postulated to be involved in mediating the toxicity of Cry1Ab. The cDNA encoding the 210?kDa protein, termed BT-R₁ (Bacillus thuringiensis receptor-1), was recently cloned, and shows limited homology to the cadherin superfamily of proteins. Quite naturally, there is a great deal of interest in the characterization of BT-R ₁ , the gene encoding the 210?kDa Cry1Ab binding protein. The studies presented here involve the use of various restriction fragments prepared from the cDNA encoding BT-R₁ as probes of Southern blots bearing M. sexta genomic DNA cleaved with a variety of restriction endonucleases. These Southern blot data reveal that there are two discrete regions within the M. sexta genome which encode sequences homologous to BT-R₁. On the basis of the signal intensities seen on Southern blots, it appears that only one of these genes encodes BT-R₁, whereas the other is a closely related homologue.     

Preparation of chloroplast DNA from pea plastids isolated in a medium of high ionic strength

A simple, rapid, and inexpensive method for the preparation and purification of chloroplast DNA (cpDNA) from pea has been developed. The crucial step is the isolation of chloroplasts in a medium of high ionic strength (I congruent equal to 1.40 M). CpDNA from pea prepared according to this method has successfully been used for restriction enzyme mapping, Southern transfers, and cloning.     

Interferon and cytotoxic factor (cytotoxin) released in the blood of mice infected with Mycobacterium bovis BCG. III. Interferon and cytotoxin induced by the specific antigen as compared with those induced by bacterial lipopolysaccharide

The time course of development and decline of the ability of BCG-infected mice to produce interferon in the serum in response to the intravenous infection of purified protein derivative of tuberculin (PPD) was very similar to that of their systemic hypersensitivity to PPD. A cytotoxic factor (cytotoxin) was produced in parallel with interferon in the serum of BCG-infected mice after stimulation with PPD. The duration of the period in which cytotoxin-production responsiveness to PPD was definitely detectable was much shorter than that for interferon-production responsiveness although the periods for the maximum production of interferon and cytotoxin coincided. The kinetics of release of interferon in the serum of BCG-infected mice after stimulation with PPD did not parallel that of release of cytotoxin. The four kinds of activities, interferons and cytotoxins induced by PPD and lipopolysaccharide (LPS) in the serum of BCG-infected mice, were compared for their stability to heating at 56 C and to treatment at pH 2. The kinetics of inactivation of these four activities differed significantly, when the serum was either heated at 56 C or treated at pH 2. Interferon produced in response to LPS could be neutralized by anti-L cell(NDV) interferon rabbit serum as easily as L cell (NDV) interferon, 16 times as much antiserum was required to neutralize the same amount of interferon in response to PPD, but cytotoxins induced by PPD and LPS were not neutralized at all by the antiserum. From these findings it is thought likely that interferons and cytotoxins induced by PPD and LPS in the serum of BCG-infected mice are different substances, although the antigenic relationship between cytotoxins induced by PPD and LPS remains unknown.     

The N terminus of the cardiac L-type Ca(2+) channel alpha(1C) subunit. The initial segment is ubiquitous and crucial for protein kinase C modulation, but is not directly phosphorylated.

The first 46 amino acids (aa) of the N terminus of the rabbit heart (RH) L-type cardiac Ca(2+) channel alpha(1C) subunit are crucial for the stimulating action of protein kinase C (PKC) and also hinder channel gating (Shistik, E., Ivanina, T., Blumenstein, Y., and Dascal, N. (1998) J. Biol. Chem. 273, 17901-17909). The mechanism of PKC action and the location of the PKC target site are not known. Moreover, uncertainties in the genomic sequence of the N-terminal region of alpha(1C) leave open the question of the presence of RH-type N terminus in L-type channels in mammalian tissues. Here, we demonstrate the presence of alpha(1C) protein containing an RH-type initial N-terminal segment in rat heart and brain by using a newly prepared polyclonal antibody. Using deletion mutants of alpha(1C) expressed in Xenopus oocytes, we further narrowed down the part of the N terminus crucial for both inhibitory gating and for PKC effect to the first 20 amino acid residues, and we identify the first 5 aa as an important determinant of PKC action and of N-terminal effect on gating. The absence of serines and threonines in the first 5 aa and the absence of phosphorylation by PKC of a glutathione S-transferase-fusion protein containing the initial segment suggest that the effect of PKC does not arise through a direct phosphorylation of this segment. We propose that PKC acts by attenuating the inhibitory action of the N terminus via phosphorylation of a remote site, in the channel or in an auxiliary protein, that interacts with the initial segment of the N terminus.     

Evaluation of laser-Doppler perfusion imaging for measurement of blood flow in cortical bone.

Most techniques currently available to measure blood flow in bone are time consuming and require destruction of the tissue, but laser-Doppler technology offers a less invasive method. This study assessed the utility of laser-Doppler perfusion imaging (LDI) to measure perfusion in cortical bone. Twelve mature New Zealand White rabbits were assigned to one of three groups: normal control, constriction (norepinephrine), or dilatation (nitroprusside). The left and right medial tibiae were consecutively scanned at red (634-nm) and near-infrared (810-nm) wavelengths to examine the repeatability of LDI output. The pharmacological intervention groups were injected with the respective drug, and LDI measurements at 810 nm were obtained concurrently with colored microsphere-determined flow in all of the groups. LDI effectively quantified blood flow in cortical bone and detected physiologically induced changes in perfusion. A significant positive correlation was found between microsphere-determined flow and LDI output (r = 0.6, P < 0.05). Repeatability of consecutive LDI measurements was within 5%. The effectiveness of LDI to measure perfusion in bone suggests this method has potential for investigating the role of blood flow in bone metabolism and remodeling.