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Biomedical subjects

R H Berg

Publications and source records attributed to R H Berg.

At least 19 recordsLinked to original sources
PubMed2004

Coat protein regulates formation of replication complexes during tobacco mosaic virus infection.

The genome of tobacco mosaic virus (TMV) encodes replicase protein(s), movement protein (MP), and capsid protein (CP). On infection, one or more viral proteins direct the assembly of virus replication complexes (VRCs), in association with host-derived membranes. The impact of CP-mediated resistance on the structures of the replication complexes was examined in nontransgenic and transgenic BY-2 cell lines that produce wild-type CP, mutant CP(T42W), and Ds-Red, which was targeted to endoplasmic reticulum by using immunofluorescence and 3D microscopy. We developed a model of VRCs that shows a clear association of MP with and surrounding the endoplasmic reticulum. Replicase is located within the MP bodies, as well as isolated sites throughout the cell. CP surrounds the VRCs. CP enhances the production of MP and increases the size of the VRC; however, the mutant CP(T42W) reduces the amount of MP and interferes with the formation of VRCs. We propose a regulatory role of the CP in the establishment of the VRC. We suggest that the lack of formation of VRCs restricts the efficiency of virus replication and the formation of virus movement complexes, resulting in restriction of cell-cell spread of infection. This results in higher levels of plant CP-mediated protection provided by CP(T42W).

Capsid Proteins↗
PubMed2004

Evaluation of spectral imaging for plant cell analysis.

Fluorescence imaging at high spectral resolution is now a practical reality and has great promise in plant cell biology. Emission spectral curve data can be used computationally to distinguish spectrally similar fluorophores, or to remove autofluorescence, and to spectrally analyse autofluorescent molecules, which are especially abundant in plant tissues. Examples of these applications in plant cells are given, and a comparison is made between the current offerings in spectral imaging laser scanning confocal microscopes.

Fluorescent Dyes↗
PubMed2000

Structural properties of DNO investigated with pyrene excimer formation.

Four diamino acid-Nalpha-substituted oligopeptide (DNO) oligomers substituted with pyrenyl as photophysical probes were synthesized. The excimer formation and ground-state association of the pyrenyl groups were investigated by means of absorption and steady-state fluorescence spectroscopy together with time-resolved fluorescence techniques. The photophysical parameters obtained from the different derivatives reflect the secondary structural properties of the DNO backbones.

Oligopeptides↗
PubMed1994

Evidence of a myoepithelial cell in tick salivary glands.

Type III acini from feeding female Dermacentor variabilis varied in size during in vitro and in vivo fluid production. As the type III acinus enlarged, its lumen enlarged and the adlumenal cell became thinner. As the acinus contracted, its lumen became smaller while the adlumenal cell became wider. Actin was demonstrated in salivary glands using an immunoblot technique. Actin was localized in the adlumenal cells of type III acini with fluorescent microscopy using rhodamine-phalloidin and with electron microscopy using heavy meromyosin to decorate actin filaments. Pre-treatment of salivary glands with cytochalasin D abolished fluorescence in adlumenal cells subsequently treated with rhodamine-phalloidin. These results support the hypothesis that the adlumenal cell in type III acini functions as a myoepithelial cell.

Actins↗
PubMed1993

Single base pair mutation analysis by PNA directed PCR clamping.

A novel method that allows direct analysis of single base mutation by the polymerase chain reaction (PCR) is described. The method utilizes the finding that PNAs (peptide nucleic acids) recognize and bind to their complementary nucleic acid sequences with higher thermal stability and specificity than the corresponding deoxyribooligonucleotides and that they cannot function as primers for DNA polymerases. We show that a PNA/DNA complex can effectively block the formation of a PCR product when the PNA is targeted against one of the PCR primer sites. Furthermore, we demonstrate that this blockage allows selective amplification/suppression of target sequences that differ by only one base pair. Finally we show that PNAs can be designed in such a way that blockage can be accomplished when the PNA target sequence is located between the PCR primers.

Alleles↗
PubMed1993

PNA hybridizes to complementary oligonucleotides obeying the Watson-Crick hydrogen-bonding rules.

DNA analogues are currently being intensely investigated owing to their potential as gene-targeted drugs. Furthermore, their properties and interaction with DNA and RNA could provide a better understanding of the structural features of natural DNA that determine its unique chemical, biological and genetic properties. We recently designed a DNA analogue, PNA, in which the backbone is structurally homomorphous with the deoxyribose backbone and consists of N-(2-aminoethyl)glycine units to which the nucleobases are attached. We showed that PNA oligomers containing solely thymine and cytosine can hybridize to complementary oligonucleotides, presumably by forming Watson-Crick-Hoogsteen (PNA)2-DNA triplexes, which are much more stable than the corresponding DNA-DNA duplexes, and bind to double-stranded DNA by strand displacement. We report here that PNA containing all four natural nucleobases hybridizes to complementary oligonucleotides obeying the Watson-Crick base-pairing rules, and thus is a true DNA mimic in terms of base-pair recognition.

Base Sequence↗
PubMed1993

DNA unwinding upon strand-displacement binding of a thymine-substituted polyamide to double-stranded DNA.

It was recently found that polyamide nucleic acid (PNA) analogues consisting of thymines attached to an aminoethylglycine backbone bind strongly and sequence-selectively to adenine sequences of oligonucleotides and double-stranded DNA [Nielsen, P. E., Egholm, M., Berg, R. H. & Buchardt, O. (1991) Science 254, 1497-1500]. It was concluded that the binding to double-stranded DNA was accomplished via strand displacement, in which the PNA bound to the Watson-Crick complementary adenine-containing strand, whereas the thymine-containing strand was extruded in a virtually single-stranded conformation. This model may provide a general way in which to obtain sequence-specific recognition of any sequence in double-stranded DNA by Watson-Crick hydrogen-bonding base-pair recognition, and it is thus paramount to rigorously establish this binding mode for synthetic DNA-binding ligands. We now report such results from electron microscopy. Furthermore, we show that binding of PNA to closed circular DNA results in unwinding of the double helix corresponding to approximately one turn of the double helix per 10 base pairs. The DNA.PNA complex, which is formed at low salt concentration (only a small portion of DNA molecules show complex formation at NaCl concentration higher than 40 mM), is exceptionally kinetically stable and cannot be dissociated by increasing salt concentration up to 500 mM.

DNA↗
PubMed1993

Sequence specific inhibition of DNA restriction enzyme cleavage by PNA.

Plasmids containing double-stranded 10-mer PNA (peptide nucleic acid chimera) targets proximally flanked by two restriction enzyme sites were challenged with the complementary PNA or PNAs having one or two mismatches, and the effect on the restriction enzyme cleavage of the flanking sites was assayed. The following PNAs were used: T10-LysNH2, T5CT4-LysNH2 and T2CT2CT4-LysNH2 and the corresponding targets cloned into pUC 19 were flanked by BamH1, Sal1 or Pstl sites, respectively. In all cases it was found that complete inhibition of restriction enzyme cleavage was obtained with the complementary PNA, a significantly reduced effect was seen with a PNA having one mismatch, and no effect was seen with a PNA having two mismatches. These results show that PNA can be used as sequence specific blockers of DNA recognizing proteins.

Base Sequence↗
PubMed1993

Peptide nucleic acids and their potential applications in biotechnology.

Peptide nucleic acids (PNAs) are novel DNA mimics in which the sugar-phosphate backbone has been replaced with a backbone based on amino acids. PNAs exhibit sequence-specific binding to DNA and RNA with higher affinities and specificities than unmodified DNA. They are resistant to nuclease and protease attack in serum and cellular extracts and, thus, appear very promising as diagnostic and biomolecular probes, and possibly as antisense and antigene drugs.

Biotechnology↗
PubMed1993

Peptide nucleic acids (PNAs): potential antisense and anti-gene agents.

The binding of peptide nucleic acids (PNAs) T10-LysNH2, T5CT4-LysNH2 and T2CT2CT4-LysNH2 to double-stranded DNA targets A10, A5GA4 and A2GA2GA4 was studied by nuclease S1 probing. It is found that the PNAs bind preferentially to their complementary targets, weaker to targets containing one mismatch and not to targets containing two mismatches. Using an RNA polymerase T3 in vitro transcription system, it is found that a PNA T10-LysNH2 bound downstream from the promoter causes transcription elongation arrest at the PNA binding site only when the PNA is bound to the template strand. Finally, it is shown that primer extension by Taq DNA polymerase on a single-stranded template is arrested at an occupied PNA T10 binding site. These results are discussed in relation to PNAs as potential anti-sense and anti-gene drugs.

Base Sequence↗
PubMed1992

Induction of Pseudoactinorhizae by the Plant Pathogen Agrobacterium rhizogenes.

Infection of Elaeagnus angustifolia cotyledonary wounds by Agrobacterium rhizogenes strain NCPPB 2659 resulted in the formation of pseudoactinorhizae on roots differentiated from callus. These pseudoactinorhizal root nodules were anatomically indistinguishable from the actinorhizae induced by the plant's microsymbiont Frankia. This unusual hairy root phenotype provides support for the concept that the genetic program for actinorhiza morphogenesis resides in the plant's genome.

Journal Article↗
PubMed1991

Sequence-selective recognition of DNA by strand displacement with a thymine-substituted polyamide.

A polyamide nucleic acid (PNA) was designed by detaching the deoxyribose phosphate backbone of DNA in a computer model and replacing it with an achiral polyamide backbone. On the basis of this model, oligomers consisting of thymine-linked aminoethylglycyl units were prepared. These oligomers recognize their complementary target in double-stranded DNA by strand displacement. The displacement is made possible by the extraordinarily high stability of the PNA-DNA hybrids. The results show that the backbone of DNA can be replaced by a polyamide, with the resulting oligomer retaining base-specific hybridization.

Base Sequence↗
PubMed1991

Effect of antitumor diarylsulfonylureas on in vivo and in vitro mitochondrial structure and functions.

Diarylsulfonylureas are novel oncolytic agents shown to have therapeutic activity against both rodent solid tumors and xenografts of human tumors in mice. Previous studies have shown that diarylsulfonylureas localize in mitochondria and cause morphological changes in these organelles. We have investigated the mechanism of action of diarylsulfonylureas, namely, N-(5-indanylsulfonyl)-N'-(4-chlorophenyl)urea (ISCU) and the N-4-methyl analogue (MPCU), by studying their effect on mitochondrial morphology and uptake of rhodamine 123 in GC3/c1 cells in culture and the oxidative phosphorylation in isolated mitochondria from mouse liver, using pyruvate-malate and succinate as substrates. Morphometric analysis of mitochondria in GC3/c1 cells exposed to ISCU showed that ISCU (165 microM) doubled the mitochondrial size after 24-h exposure in culture. Also, ISCU (100 microM), like 40 microM carbonylcyanide p-trifluoromethoxyphenylhydrazone, significantly reduced the rhodamine 123 uptake by GC3/c1 cells studied by flow cytometry. In isolated mitochondria both ISCU and MPCU uncoupled oxidative phosphorylation at 50 microM, with pyruvate-malate as substrate, as was indicated by a significant increase in the State 4 oxygen consumption. This resulted in the loss of ADP phosphorylation and, therefore, the ADP/oxygen ratio was reduced to zero and the respiratory control ratio to one. The succinate oxidation was also significantly impaired by ISCU, causing some decrease in ADP phosphorylation. On the other hand, MPCU did not exhibit any significant effect on the oxidation of succinate. At concentrations of lower than 50 microM, both of these compounds exhibited a deleterious effect, causing damage to mitochondrial functions in the presence of pyruvate-malate as substrates. These data confirm, through morphometric analysis, our previous qualitative observations of abnormal mitochondrial morphology observed in GC3/c1 cells grown in the presence of high concentrations of ISCU and MPCU and further suggest that diarylsulfonylureas, by uncoupling mitochondrial oxidative phosphorylation, may lower cellular ATP. It is probable that this mechanism contributes, at least partially, to cytotoxicity in GC3/c1 cells exposed to high concentrations of ISCU for relatively brief periods (2 to 4 h) and possibly contributes to cytotoxicity at drug concentrations that can be achieved in rodents.

Adenosine Diphosphate↗
PubMed1991

Murine T cell responses to melittin and its analogs.

The 26-residue peptide melittin from bee venom elicits high IgG1 and IgE responses in selected strains of mice. The antibody responses were shown previously to be specific mainly for the region of residue 20-26. The T cell epitope of melittin in H-2d-restricted mice is now found to be primarily in residue 11-19, corresponding to an alpha-helical amphiphilic segment of the molecule. Melittin-specific T cell lines have varying responses to different structural analogs of the melittin T cell epitope, and the results indicate that the antigenicity of T cell epitope peptides depend more on their primary structure than on their secondary structure. Melittin-specific T cell clones are found to be CD4+ and secrete IL-4, and are restricted to presentation on I-Ad or I-Ed. The I-Ad- or I-Ed-restricted clones differ in their responses to different analogs of melittin.

Amino Acid Sequence↗
PubMed1988

Enzyme-gold affinity labelling of cellulose.

The enzyme-linked colloidal gold affinity labelling technique was tested as a method to localize cellulose on thin sections of plant cell walls and slime mold spores. Commercially available cellulase from cultures of Trichoderma reesei, the main components being cellobiohydrolase I and II (CBH I, CBH II) and endoglucanase (EG), was linked to colloidal gold by using standard techniques and applied as a dilute, buffered suspension to thin sections. After brief exposure, e.g., 15-30 minutes, cellulose exposed on the surface of sections was labelled with the enzyme-gold complex. Poststaining did not appear to have a deleterious effect on the labelled sections. The specificity of labelling was demonstrated by its complete inhibition when carboxymethylcellulose was incorporated in the labelling mixture, by lack of labelling of 1,4-beta-mannans or 1,3-beta-xylans in noncellulosic walls of marine algae, by lack of labelling of 1,4-beta-glucans in chitin, by much lower labelling density when done at 4 degrees C, and by lack of labelling when sections were predigested with cellulase. Labelling with the crude commercial cellulase was compared to labelling with purified CBH I-, CBH II-, and EG-linked colloidal gold, and the labelling pattern was similar. This method was found useful on conventionally fixed material and required no special preparation other than the use of inert (Ni or Au) grids and 0.5% gelatin to reduce nonspecific binding of the gold complex. Labelling was similar in the several embedding resins tested: LR White, Lowicryl K4M, Epon 812, and Spurr's.(ABSTRACT TRUNCATED AT 250 WORDS)

Affinity Labels↗
PubMed1980

Biology of azospirillum-sugarcane association: enhancement of nitrogenase activity.

Azospirillum brasilense was reisolated from associations with callus tissue cultures of sugarcane and compared with stock cultures of the inoculated bacterium and related strains. Although the reisolate had a growth rate similar to stock cultures, it exhibited a severalfold increase in maximum specific activity of nitrogenase. The reisolate and the parent culture had similar ultrastructure. The general ultrastructure of Azospirillum is described. The bacterium was capsulated when grown on nitrogen-free nutrient agar plates and on callus, but was not capsulated when growing in a subsurface zone in N-free semisolid nutrient agar, except rarely in aging cultures. Capsulation may be a protective mechanism against unfavorable pO(2) under dinitrogen-fixing conditions. Pleomorphism occurred in capsulated forms, and the ultrastructure of these forms is described.

Journal Article↗
PubMed1969

Pot and booze.

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Cannabis↗