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Biomedical subjects

P Elias

Publications and source records attributed to P Elias.

At least 19 recordsLinked to original sources
PubMed2004

A new probiotic cheese with antioxidative and antimicrobial activity.

The aim of our study was to develop an original probiotic cheese based on the Estonian open-texture, smear-ripened, semisoft cheese "Pikantne." Cheese was produced by two methods using cheese starter cultures (Probat 505) in combination with 0.04% of probiotic Lactobacillus fermentum strain ME-3 (10(9) cfu/mL) with high antimicrobial activity and antioxidative properties. The probiotic Lactobacillus was added into milk simultaneously with starter cultures (cheese A) and into drained curd (cheese B). After addition of probiotic L. fermentum ME-3, the cheese composition, flavor, and aroma were comparable to the control cheese (score values = 4.5, 4.2, and 3.7 for control cheese, cheese A, and cheese B, respectively). Cheese A, which had good sensory properties, was chosen for further testing of viability and probiotic properties. The probiotic strain was found to withstand the technological processing of cheese, surviving and sustaining moderate antimicrobial and high antioxidative activity throughout ripening and storage (the ripened cheese contained approximately 5 x 10(7) cfu/g viable ME-3 cells), although the viability of the ME-3 strain incorporated into the cheese showed a slight decrease between d 24 and 54 after cheese preparation. Semisoft cheese "Pikantne" serves as a suitable carrier of antimicrobial and antioxidative L. fermentum ME-3.

Anti-Infective Agents↗
PubMed2001

Complementary intrastrand base pairing during initiation of Herpes simplex virus type 1 DNA replication.

The herpes simplex virus type 1 origin of DNA replication, oriS, contains three copies of the recognition sequence for the viral initiator protein, origin binding protein (OBP), arranged in two palindromes. The central box I forms a short palindrome with box III and a long palindrome with box II. Single-stranded oriS adopts a conformation, oriS*, that is tightly bound by OBP. Here we demonstrate that OBP binds to a box III-box I hairpin with a 3' single-stranded tail in oriS*. Mutations designed to destabilize the hairpin abolish the binding of OBP to oriS*. The same mutations also inhibit DNA replication. Second site complementary mutations restore binding of OBP to oriS* as well as the ability of mutated oriS to support DNA replication. OriS* is also an efficient activator of the hydrolysis of ATP by OBP. Sequence analyses show that a box III-box I palindrome is an evolutionarily conserved feature of origins of DNA replication from human, equine, bovine, and gallid alpha herpes viruses. We propose that oriS facilitates initiation of DNA synthesis in two steps and that OBP exhibits exquisite specificity for the different conformations oriS adopts at these stages. Our model suggests that distance-dependent cooperative binding of OBP to boxes I and II in duplex DNA is succeeded by specific recognition of a box III-box I hairpin in partially unwound DNA.

Adenosine Triphosphate↗
PubMed2001

Rep-dependent initiation of adeno-associated virus type 2 DNA replication by a herpes simplex virus type 1 replication complex in a reconstituted system.

Productive infection by adeno-associated virus type 2 (AAV) requires coinfection with a helper virus, e.g., adenovirus or herpesviruses. In the case of adenovirus coinfection, the replication machinery of the host cell performs AAV DNA replication. In contrast, it has been proposed that the herpesvirus replication machinery might replicate AAV DNA. To investigate this question, we have attempted to reconstitute AAV DNA replication in vitro using purified herpes simplex virus type 1 (HSV-1) replication proteins. We show that the HSV-1 UL5, UL8, UL29, UL30, UL42, and UL52 gene products along with the AAV Rep68 protein are sufficient to initiate replication on duplex DNA containing the AAV origins of replication, resulting in products several hundred nucleotides in length. Initiation can occur also on templates containing only a Rep binding site and a terminal resolution site. We further demonstrate that initiation of DNA synthesis can take place with a subset of these factors: Rep68 and the UL29, UL30, and UL42 gene products. Since the HSV polymerase and its accessory factor (the products of the UL30 and UL42 genes) are unable to efficiently perform synthesis by strand displacement, it is likely that in addition to creating a hairpin primer, the AAV Rep protein also acts as a helicase for DNA synthesis. The single-strand DNA binding protein (the UL29 gene product) presumably prevents reannealing of complementary strands. These results suggest that AAV can use the HSV replication apparatus to replicate its DNA. In addition, they may provide a first step for the development of a fully reconstituted AAV replication assay.

DNA Helicases↗
PubMed2000

Recombination during early herpes simplex virus type 1 infection is mediated by cellular proteins.

Homologous recombination was examined in cells infected with herpes simplex virus type I. Circular and linear DNA with directly repeated sequences was introduced as recombination substrates into cells. Recombination was measured either by origin-dependent amplification of recombination products or by recombination-dependent expression of luciferase from a disrupted gene. Homologous recombination in baby hamster kidney cells converted linear DNA to circular templates for DNA replication and luciferase expression in the complete absence of virus. The products of homologous recombination were efficiently amplified by the viral replication apparatus. The efficiency of recombination was dependent on the structure of the substrate as well as the cell type. Linear DNA with the direct repeats at internal positions failed to recombine in Balb/c 3T3 cells and induced p53-dependent apoptosis. In contrast, linear DNA with directly repeated sequences precisely at the ends recombined and replicated in 3T3 cells. Homologous recombination in baby hamster kidney cells did not depend on the position of the repeated sequences. We conclude that homologous recombination is independent of viral gene functions and that it is likely to be carried out by cellular proteins. We suggest that homologous recombination between directly repeated sequences in the linear herpes simplex virus type 1 chromosome may help to avoid p53-dependent apoptosis and to promote viral DNA replication.

3T3 Cells↗
PubMed2000

Leading and lagging strand DNA synthesis in vitro by a reconstituted herpes simplex virus type 1 replisome.

The synthesis of double-stranded DNA by a rolling circle mechanism was reconstituted in vitro with a replisome consisting of the DNA polymerase-UL42 complex and the heterotrimeric helicase-primase encoded by herpes simplex virus type 1. Okazaki fragments 3 kilobases in length and leading strands that may exceed 10 kilobases are produced. Lagging strand synthesis is stimulated by ribonucleoside triphosphates. DNA replication appears to be processive because it resists competition with an excess of (dT)(150)/(dA)(20). The single-strand DNA binding protein ICP8 is not required, and high concentrations of ICP8 can, in fact, inhibit lagging strand synthesis. The inhibition can, however, be overcome by the addition of an excess of the UL8 component of the helicase-primase. Rolling circle replication by the herpesvirus and bacteriophage T7 replisomes appears to proceed by a similar mechanism.

DNA↗
PubMed2000

A novel conformation of the herpes simplex virus origin of DNA replication recognized by the origin binding protein.

The Herpes simplex virus type I origin binding protein (OBP) is a sequence-specific DNA-binding protein and a dimeric DNA helicase encoded by the UL9 gene. It is required for the activation of the viral origin of DNA replication oriS. Here we demonstrate that the linear double-stranded form of oriS can be converted by heat treatment to a stable novel conformation referred to as oriS*. Studies using S1 nuclease suggest that oriS* consists of a central hairpin with an AT-rich sequence in the loop. Single-stranded oligonucleotides corresponding to the upper strand of oriS can adopt the same structure. OBP forms a stable complex with oriS*. We have identified structural features of oriS* recognized by OBP. The central oriS palindrome as well as sequences at the 5' side of the oriS palindrome were required for complex formation. Importantly, we found that mutations that have been shown to reduce oriS-dependent DNA replication also reduce the formation of the OBP-oriS* complex. We suggest that oriS* serves as an intermediate in the initiation of DNA replication providing the initiator protein with structural information for a selective and efficient assembly of the viral replication machinery.

Base Sequence↗
PubMed1998

The herpes simplex virus type 1 helicase-primase. Analysis of helicase activity.

The rate of unwinding of duplex DNA by the herpes simplex virus type 1 (HSV-1)-encoded helicase-primase (primosome) was determined by measuring the rate of appearance of single strands from a circular duplex DNA containing a 40-nucleotide 5' single-stranded tail, i.e. a preformed replication fork, in the presence of the HSV-1 single strand DNA-binding protein, infected cell protein 8 (ICP8). With this substrate, the rate at low ionic strength was highly sensitive to Mg2+ concentration. The Mg2+ dependence was a reflection of both the requirement for ICP8 for helicase activity and the ability of ICP8 to reverse the helicase reaction as a consequence of its capacity to anneal homologous single strands at Mg2+ concentrations in excess of 3 mM. The rate of unwinding of duplex DNA by the HSV-1 primosome was also determined indirectly by measuring the rate of leading strand synthesis with a preformed replication fork as template in the presence of the T7 DNA polymerase. The value of 60-65 base pairs unwound/s by both methods is consistent with the rate of 50 base pairs/s estimated for the rate of fork movement in vivo during replication of pseudorabies virus, another herpesvirus. Interaction with the helicase-primase did not increase its helicase activity.

Base Sequence↗
PubMed1998

The herpes simplex virus type 1 origin binding protein. Specific recognition of phosphates and methyl groups defines the interacting surface for a monomeric DNA binding domain in the major groove of DNA.

The UL9 gene of herpes simplex virus type 1 (HSV-1) encodes an origin binding protein (OBP). It is an ATP-dependent DNA helicase and a sequence-specific DNA-binding protein. The latter function is carried out by the C-terminal domain of OBP (DeltaOBP). We have now performed a quantitative analysis of the interaction between DeltaOBP and its recognition sequence, GTTCGCAC, in oriS. Initially optimal conditions for binding were carefully determined. We observed that complexes with different electrophoretic mobilities were formed. A cross-linking experiment demonstrated that nonspecific complexes containing 2 or more protein monomers per DNA molecule were formed at high protein concentrations. The specific complex formed at low concentrations of DeltaOBP had an electrophoretic mobility corresponding to a 1:1 complex. We then demonstrated that the methyl groups of thymine in the major groove were essential for high affinity binding. Changes in the minor groove had considerably smaller effects. Ethylation interference experiments indicated that specific contacts were made between OBP and three phosphates in the recognition sequence. Finally, these observations were used to present a model of the surface of DNA that interacts with DeltaOBP in a sequence-specific manner.

Base Sequence↗
PubMed1998

Acute barrier perturbation abolishes the Ca2+ and K+ gradients in murine epidermis: quantitative measurement using PIXE.

Epidermal permeability barrier homeostasis requires the delivery of lipids and hydrolytic enzymes by lamellar body exocytosis from the uppermost granular cells, a process that is upregulated following barrier disruption. As lamellar body secretion is controlled by ionic concentrations, especially Ca2+ and K+, we used a quantitative technique, microbeam proton-induced X-ray emission, to measure Ca2+, K+, Cl-, and P concentrations before and after acute barrier perturbation by acetone applications. We found a steep gradient of Ca2+ in normal tissue, peaking in the outer stratum granulosum, which disappeared after barrier disruption, and partially reformed as the barrier recovered. A similar gradient, peaking somewhat lower in the epidermis (i.e., at the stratum granulosum-stratum corneum interface), was found for K+. Epidermal concentrations of K+ also decreased after barrier abrogation, although to a lesser extent than Ca2+. In contrast, P and Cl- demonstrated distribution gradients at baseline, which remained unchanged after barrier disruption. These studies quantitate the levels of Ca2+, K+, Cl-, and P within specific epidermal cell layers at baseline, and in relation to changes in permeability barrier integrity. Ca2+ and K+, but not Cl- or P, decrease after barrier disruption, consistent with these two ion's role in barrier repair.

Animals↗
PubMed1997

The UL8 subunit of the heterotrimeric herpes simplex virus type 1 helicase-primase is required for the unwinding of single strand DNA-binding protein (ICP8)-coated DNA substrates.

The Herpes simplex virus type 1 primosome consists of three subunits that are the products of the UL5, UL8, and UL52 genes. The heterotrimeric enzyme has DNA-dependent ATPase, helicase, and primase activities. Earlier studies show that a subassembly consisting of the UL5 and UL52 gene products was indistinguishable from the heterotrimeric enzyme in its helicase and primase activities. We demonstrate here that the UL8 protein is required for the helicase activity of the UL5/52 subassembly on long duplex DNA substrates (>30 nucleotides) with a single-stranded DNA loading site fully coated with the virus-encoded single strand DNA binding protein, ICP8. The Escherichia coli single strand DNA binding protein cannot substitute for ICP8, suggesting a specific physical interaction between ICP8 and the UL8 protein. Surface plasmon resonance measurements demonstrated an interaction between ICP8 and the UL5/52/8 heterotrimer but not with the UL5/52 subassembly or the UL8 protein alone. At a subsaturating level of ICP8, the UL5/52 subassembly does show helicase activity, suggesting that the subassembly can bind to single-stranded DNA but not to ICP8-coated DNA.

Animals↗
PubMed1997

Herpes simplex virus: selection of origins of DNA replication.

A selection procedure was devised to study the role of cis -acting sequences at origins of DNA replication. Two regions in Herpes simplex virus oriS were examined: an AT-rich spacer sequence and a putative binding site, box III, for the origin binding protein. Plasmid libraries were generated using oligonucleotides with locally random sequences. The library, amplified in Escherichia coli , was used to transfect BHK cells followed by superinfection with HSV-1. Replicated plasmids resistant to Dpn I cleavage were amplified in E. coli. The selection scheme was repeated. Plasmids were isolated at different stages of the procedure and their replication efficiency was determined. Efficiently replicating plasmids had a high AT content in the spacer sequence as well as a low helical stability of this region. In contrast, this was not seen using the box III library. We also noted that the wild type sequence invariably dominated the library after five rounds of selection. These plasmids arose from recombination between plasmids and viral DNA. Our results imply that a large group of sequences can mechanistically serve as origins of DNA replication. In a viral system, however, where the initiation process might be rate-limiting, this potentially large group of sequences would always converge towards the most efficient replicator.

Animals↗
PubMed1997

TRF1 is a dimer and bends telomeric DNA.

TRF1 is a mammalian telomeric protein that binds to the duplex array of TTAGGG repeats at chromosome ends. TRF1 has homology to the DNA-binding domain of the Myb family of transcription factors but, unlike most Myb-related proteins, TRF1 carries one rather than multiple Myb-type DNA-binding motifs. Here we show that TRF1 binds DNA as a dimer using a large conserved domain near the N-terminus of the protein for TRF1-TRF1 interactions. Dimerization was observed both in a complex with DNA and in the yeast two-hybrid assay. TRF1 dimers were found to require both Myb repeats for the formation of a stable complex with DNA, indicating a parallel between the DNA-binding mode of TRF1 and other Myb-related proteins. TRF1 was found to have a number of biochemical similarities to Rap1p, a distantly related DNA-binding protein that functions at telomeres in yeast. Rap1p and TRF1 both require two Myb motifs for DNA binding and both factors bind along their cognate telomeric sequences without showing strong cooperative interactions between adjacent proteins. Furthermore, TRF1 was found to bend its telomeric site to an angle of -120 degrees. Since Rap1p similarly distorts telomeric DNA, we propose that DNA bending is important for the function of telomeres in yeast and mammals.

Amino Acid Sequence↗
PubMed1997

Direct repeats of the herpes simplex virus a sequence promote nonconservative homologous recombination that is not dependent on XPF/ERCC4.

We have examined mechanisms of recombination in mammalian cells infected with herpes simplex virus type 1 (HSV-1). Amplification of plasmids containing a viral origin of replication, oriS, in cells superinfected with HSV-1 revealed that linear DNA could be efficiently converted to templates for replication. Two distinct pathways were observed: imprecise end joining and nonconservative homologous recombination. We noted that direct repeats of the viral a sequence promoted efficient nonconservative homologous recombination in BHK cells as well as human repair-proficient 1BR.3N cells and xeroderma pigmentosum group F (XP-F) cells. The reaction gave rise to functional a sequences supporting the formation of defective viruses. It did not seem to proceed by single-strand annealing since it occurred in the absence of XPF/ERCC4, the mammalian homolog of the Rad1 endonuclease from Saccharomyces cerevisiae. In contrast, direct repeats of a 161-bp nonviral sequence did not take part in nonconservative homologous recombination in XP-F cells. Our results suggest that homologous recombination may be involved in the circularization of viral genomes. Furthermore, they demonstrate that amplification of recombination products supported by HSV-1 allows a direct examination of pathways for double-strand-break repair in human cells.

Animals↗
PubMed1996

Characterization of a binding site for the herpes simplex virus type 1 UL9 origin-binding protein within the UL9 gene.

Gene UL9 of herpes simplex virus type 1 (HSV-1) encodes a sequence-specific origin-binding protein (OBP) that plays a direct and essential role in viral DNA synthesis. A search of the complete HSV-1 genomic sequence for possible OBP binding sites lying outside the known origins of replication revealed the presence of a very close match to the OBP recognition sequence within the UL9 coding region. The ability of OBP to bind to this site (referred to as the 'UL9 box') was confirmed by DNase I footprinting and gel retardation assays, and filter binding experiments demonstrated that the affinity of OBP for the UL9 box was of the same order as for its high affinity sites within the three replication origins. To investigate whether binding of OBP to the UL9 box played a role during viral replication we constructed a mutant virus in which the sequence was altered in such a way as to preserve the encoded amino acid sequence whilst abolishing the ability of OBP to bind. Growth of the virus was indistinguishable from wild-type and no alterations were observed in the accumulation of transcripts from the UL9 region of the genome. In addition, a DNA fragment containing the UL9 box sequence did not exhibit origin activity in a transient assay for viral DNA synthesis. We therefore conclude that binding of OBP to the UL9 box is not essential for virus growth and that expression of the UL9 gene is unlikely to be autoregulated through this site.

Base Sequence↗
PubMed1996

Inhibition of topoisomerase II by ICRF-193 prevents efficient replication of herpes simplex virus type 1.

Cellular topoisomerase II is specifically inactivated by the drug ICRF-193. This compound turns topoisomerase II into a closed clamp that is unable to cleave DNA. We have investigated the effects of this inhibitor on the replication of herpes simplex virus type 1. We show that ICRF-193 at low multiplicities of infection dramatically inhibits viral DNA synthesis and the production of infectious virus. The inhibition is less efficient at high multiplicities of infection. In addition, inhibition of viral DNA synthesis was observed only when ICRF-193 was present during the first 4 h of the infectious cycle. The transient replication of plasmids containing a herpes simplex virus type 1 origin of DNA replication, oriS, was affected by ICRF-193 in the same way. In contrast, neither cellular DNA synthesis nor replication of plasmids containing a simian virus 40 origin of DNA replication was inhibited. The observed effect on herpes simplex virus DNA replication was not caused by a decreased transcription of replication genes inasmuch as the levels of UL8, UL9, UL29, and UL30 rmRNAs were unaffected by the drug. These results suggest that topoisomerase II plays a vital role during the replication of herpes simplex virus type 1 DNA. We speculate that topoisomerase II is involved in the decatenation of newly synthesized daughter molecules.

Animals↗
PubMed1995

The DNA ligands influence the interactions between the herpes simplex virus 1 origin binding protein and the single strand DNA-binding protein, ICP-8.

The herpes simplex virus type 1 (HSV-1) origin binding protein, OBP, is a DNA helicase specifically stimulated by the viral single strand DNA-binding protein, ICP-8. The stimulation is dependent on direct protein-protein interactions between the C-terminal domain of OBP, delta OBP, and ICP 8 (Boehmer, P.E., Craigie, M.C., Stow, N.D., and Lehman, I.R. (1994) J. Biol. Chem. 269, 29329-29334). We have now observed that this interaction is dramatically influenced by the nature of the DNA ligand. Stable complexes between delta OBP, ICP 8, and double-stranded DNA, presented either as a specific duplex oligonucleotide or a restriction fragment containing the HSV-1 origin of replication, oriS, can be detected by gel chromatography and gel electrophoresis. In contrast, a single-stranded oligonucleotide, oligo(dT)65, will completely disrupt the complex between delta OBP and ICP 8. We therefore suggest that the interaction between delta OBP and ICP 8 serves to position the single strand DNA-binding protein with high precision onto single-stranded DNA at a replication fork or at an origin of DNA replication.

Base Sequence↗