Vibrational circular dichroism of A-, B-, and Z-form nucleic acids in the PO2-stretching region.

Vibrational circular dichroism (VCD) spectra were measured for H2O solutions of several natural and model DNAs (single and double strands, oligomers and polymers) in the B-form, poly(dG-dC)-poly(dG-dC) in the Z-form, and various duplex RNAs in an A-form over the PO2-stretching region. Only the symmetric PO2 stretch at approximately 1075 cm-1 yields a significant intensity VCD signal. Differences of the PO2-stretching VCD spectra found for these conformational types are consistent with the spectral changes seen in the base region, but no sequence dependence was seen in contrast to VCD for base modes. The B to Z transition is accompanied by an inversion of the PO2- VCD spectra, which is characteristic of the change in the helical sense of the nucleic acid backbone. A-RNAs give rise to the same sense of couplet VCD as do B-DNAs but have a somewhat different shape because of overlapping ribose modes. These PO2- VCD spectral characteristics have been successfully modeled using simple dipole coupling calculations. The invariability of the symmetric PO2- stretching mode VCD spectra to the base sequence as opposed to that found for the C = O stretching and base deformation modes is evidence that this mode will provide a stable indication of the DNA helical sense.     

Absence of effects of low-frequency, low-amplitude magnetic fields on the properties of gramicidin A channels.

The effects of static and low-frequency magnetic fields on gramicidin A channels have been investigated using bilayer patch clamp recording and a bridge technique capable of detecting 0.3% changes in the conductance of glyceryl monooleate membranes containing many channels. In the bridge technique the conductance was assessed using 10-ms voltage pulses applied at 10 Hz. Measurements were made for LiCl, KCl, and CsCl using magnetic fields of 50, 100, 500, and 5000 microT with the frequency scanned from 10-200 Hz. The combinations of static and low-frequency fields employed include the "cyclotron resonance" conditions at which effects had been predicted to occur. In no case was there any detectable change in conductance when the magnetic fields were applied or changed. Potassium currents through single gramicidin channels have been recorded for patches in which several channels may be open at once. Fields were applied for 2 min periods interleaved with 2 min controls. Methods have been developed to analyze the multichannel records to reveal the amplitude and duration of the channels together with the frequency, depth, and apparent period of flickers. No significant differences were observed between the control and field-exposed recording periods. The peak of the distribution of opening and closing transitions always coincided for fields on and off within the resolution, 0.4%, of the recordings. There are at least two types of flicker, one with typical period less than 0.1 ms, the other with typical period from 0.3-0.8 ms. Most of the latter were not complete closures with the conductance during a flicker 15-20% above the level for a full closure.     

Variations in Immune Response of Popillia japonica and Acheta domesticus to Heterorhabditis bacteriophora and Steinernema Species

The infectivities of Steinernema carpocapsae, S. glaseri, S. scapterisci, and Heterorhabditis bacteriophora to Japanese beetle larvae, Popillia japonica, and house cricket adults, Acheta domesticus, were compared using external exposure and hemocoelic injection. Only H. bacteriophora and S. glaseri caused high P. japonica mortality after external exposure. When nematodes were injected, P. japonica had a strong encapsulation and melanization response to all species except S. glaseri. Heterorhabditis bacteriophora and S. carpocapsae were able to overcome the immune response, but S. scapterisci was not. All species except S. scapterisci were able to kill and reproduce within the host. Only S. scapterisci and S. carpocapsae caused A. domesticus mortality after external exposure. When nematodes were injected, A. domesticus had a strong immune response to all species except S. scapterisci. Steinernema carpocapsae effectively overcame the strong immune response and caused high host mortality, but S. glaseri and H. bacteriophora did not. Steinernema scapterisci caused high host mortality and reproduced, S. glaseri and H. bacteriophora caused low host mortality but only S. glaseri reproduced, and S. carpocapsae was able to kill the host but reproduced poorly. Most (ca. 90%) of the S. carpocapsae in the hemocoel of P. japonica became encapsulated and melanized within 8 hours postinjection. The symbiotic bacterium, Xenorhabduf nematophilus, was often released before this encapsulation and melanization.     

Induction of Nitric Oxide Synthase in Rat C6 Glioma Cells

Abstract: We have examined the induction of nitric oxide syhthase (NOS) activity in the rat astrocyte-derived C6 glioma cell line. In contrast to the previous results with primary astrocyte cultures, incubation of C6 cells with bacterial endotoxin lipopolysaccharide (LPS; 1 μg/ml for 24 h) did not stimulate NO₂ production. However, addition of either tumor necrosis factor-a (TNF-α) or interferon-γ (IFN-γ), cytokines that by themselves had no effect on NOS activity, imparted LPS responsiveness onto these cells in a dose-dependent manner (EC₅₀ values of 39 ng/ml of TNF-α and 9.4 U/ml of IFN-γ), and the effect of TNF-α could be further potentiated (twofold) by the presence of interleukin-1β. The simultaneous presence of TNF-α and IFN-γ yielded a greater response than either cytokine alone; however, the respective EC₅₀ values were not affected. A cytoplasmic extract from induced C6 cells catalyzed the Ca²⁺-independent conversion of l -arginine to l - citrulline, with an apparent K _m of 51.2 n M , and this activity could be blocked by l -arginine analogues in the potency order amino > methyl > nitroarginine. Immunoblot analysis revealed an apparent molecular mass of 125 kDa for the NOS protein induced in C6 cells. These results indicate that the combination of LPS plus cytokines can induce NOS activity in C6 glioma cells with properties similar to those of the enzyme expressed in primary astrocyte cultures.     

Asymmetric somatic hybridization between tall fescue (Festuca arundinacea Schreb.) and irradiated Italian ryegrass (Lolium multiflorum Lam.) protoplasts

Intergeneric asymmetric somatic hybrids have been obtained by the fusion of metabolically inactivated protoplasts from embryogenic suspension cultures ofFestuca arundinacea (recipient) and protoplasts from a non-morphogenic cell suspension ofLolium multiflorum (donor) irradiated with 10, 25, 50, 100, 250 and 500 Gy of X-rays. Regenerating calli led to the recovery of genotypically and phenotypically different asymmetric somatic hybridFestulolium plants. The genome composition of the asymmetric somatic hybrid clones was characterized by quantitative dot-blot hybridizations using dispersed repetitive DNA sequences specific to tall fescue and Italian ryegrass. Data from dot-blot hybridizations using two cloned Italian ryegrass-specific sequences as probes showed that irradiation favoured a unidirectional elimination of most or part of the donor chromosomes in asymmetric somatic hybrid clones obtained from fusion experiments using donor protoplasts irradiated at doses 250 Gy. Irradiation of cells of the donor parent with 500 Gy prior to protoplast fusion produced highly asymmetric nuclear hybrids with over 80% elimination of the donor genome as well as clones showing a complete loss of donor chromosomes. Further information on the degree of asymmetry in regenerated hybrid plants was obtained from chromosomal analysis including in situ hybridizations withL. multiflorum-specific repetitive sequences. A Southern blot hybridization analysis using one chloroplast and six mitochondrial-specific probes revealed preferentially recipient-type organelles in asymmetric somatic hybrid clones obtained from fusion experiments with donor protoplasts irradiated with doses higher than 100 Gy. It is concluded that the irradiation of donor cells before fusion at different doses can be used for producing both nuclear hybrids with limited donor DNA elimination or highly asymmetric nuclear hybrid plants in an intergeneric graminaceous combination. For a wide range of radiation doses tested (25–250Gy), the degree of the species-specific genome elimination from the irradiated partner seems not to be dose dependent. A bias towards recipient-type organelles was apparent when extensive donor nuclear genome elimination occurred.Abbreviations cpDNA Chloroplast DNA - 2, 4-D 2,4-dichlorophenoxyacetic acid - FDA fluorescein diacetate - IOA iodoacetamide - mtDNA mitochondrial DNA - RFLP restriction fragment length polymorphism     

Measurement of changes in functional muscarinic acetylcholine receptor density in single neuroblastoma cells using calcium release kinetics

Calcium release in response to the activation of muscarinic M1 and histamine H₁ receptors was studied in single N1E-115 cells using Fura-2 imaging. The objective was to relate changes in the kinetics of Ca release with reductions in functional receptor density resulting from receptor desensitization. Calcium release increased and its time course accelerated with increasing carbachol concentration with an EC₅₀ = 96 ± 8 μM. This value is similar to the binding K_D (100 μM) and the similarity shows that the activation of calcium release is limited by the number of muscarinic receptors. In contrast, the EC₅₀ for Ca release in response to histamine is 4.0 ± 0.7 μM while the binding K_D is 8.3 μM and, therefore, H₁ receptors appear to be in approximately 2-fold excess over the minimum number necessary to fully engage the Ca release mechanism.Functional surface receptor number was assayed in the population of cells by counting the total number of cells responding to agonist. A 5 min exposure to 1 mM carbachol caused 12% of cells to lose their ability to respond to carbachol, with no change in their response to histamine. Interpolating from the dose-response curve taken before desensitization, this is equivalent to an average 23% reduction in the number of muscarinic receptors. In individual cells the latency to Ca release is dose-dependent in the absence of excess receptors. The loss of functional receptors was therefore estimated from the increase in latency after desensitization, and varied from 5–48% of receptors (22 ± 18%). Muscarinic desensitization did not depend on IP₃-evoked Ca release, Ca entry, protein kinase C, NO, or cGMP. We conclude that in a population, the number of cells responding and in single cells, the latency to Ca release can serve as measures of functional receptor density.     

Differential expression inArabidopsis ofLhca2, a PSI cab gene

A cDNA clone of the geneLhca2 encoding a photosystem I (PSI) type II chlorophylla/b-binding protein was isolated fromArabidopsis thaliana. The isolation of this, the fourth PSI cab gene fromArabidopsis, confirms a previous report [1] that indicatedArabidopsis may contain all four PSI cab genes identified in other plant species.Lhca2 is a single-copy gene as are the other knownArabidopsis PSI cab genes. The patterns of developmental expression and tissue-specific regulation ofLhca2 are similar to those of other PSI and PSII cab genes, but the light induction pattern and the steady-state mRNA level ofLhca2 are distinct. This suggests that a different mechanism may be employed to regulate the expression ofLhca2.     

A conserved helical element is essential for internal initiation of translation of hepatitis C virus RNA.

Translation of hepatitis C virus (HCV) RNA is initiated by cap-independent internal ribosome binding to the 5' noncoding region (NCR). To identify the sequences and structural elements within the 5' NCR of HCV RNA that contribute to the initiation of translation, a series of point mutations was introduced within this sequence. Since the pyrimidine-rich tract is considered a characteristic feature of picornavirus internal ribosome entry site (IRES) elements, our mutational analysis focused on two putative pyrimidine tracts (Py-I and Py-II) within the HCV 5' NCR. Translational efficiency of these mutant RNAs was examined by in vitro translation and after RNA transfection into liver-derived cells. Mutational analysis of Py-I (nucleotides 120 to 130), supported by compensatory mutants, demonstrates that the primary sequence of this motif is not important but that a helical structural element associated with this region is critical for HCV IRES function. Mutations in Py-II (nucleotides 191 to 199) show that this motif is dispensable for IRES function as well. Thus, the pyrimidine-rich tract motif, which is considered as an essential element of the picornavirus IRES elements, does not appear to be a functional component of the HCV IRES. Further, the insertional mutagenesis study suggests a requirement for proper spacing between the initiator AUG and the upstream structures of the HCV IRES element for internal initiation of translation.     

Identification and characterization of a human herpesvirus 6 gene segment capable of transactivating the human immunodeficiency virus type 1 long terminal repeat in an Sp1 binding site-dependent manner.

The human immunodeficiency virus type 1 (HIV-1) long terminal repeat (LTR) is transactivated by various extracellular signals and viral cofactors that include human herpesviruses. These transactivators are capable of transactivating the HIV-1 LTR through the transactivation response element, NF-kappa B, or other regulatory binding elements. Human herpesvirus 6 (HHV-6) is a potential cofactor of HIV-1. Here, we report that an HHV-6 gene segment, ZVH14, which can neoplastically transform NIH 3T3 and human keratinocytes, is capable of transactivating HIV-1 LTR chloramphenicol acetyltransferase constructs in an Sp1 binding site-dependent manner. Transactivation increased synergistically in the presence of multiple Sp1 sites and was dramatically reduced by cotransfection with oligomers designed to form triplex structures with HIV-1 LTR Sp1 binding sites. HIV-1 LTR NF-kappa B sites were not essential for ZVH14-mediated transactivation. A putative open reading frame in ZVH14, B115, which may encode a highly basic peptide consisting of 115 amino acid residues, showed transactivation capacity similar to that of ZVH14. This open reading frame also transactivated the HIV-1 LTR in an Sp1 site-dependent fashion in African green monkey kidney cells and human T cells. These data suggest that HHV-6 may stimulate HIV-1 replication via transactivation of Sp1 binding sites present in the HIV-1 promoter.