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Biomedical subjects

A Orr

Publications and source records attributed to A Orr.

At least 37 records · Page 2Linked to original sources
PubMed1997

Replacement of the herpes simplex virus type 1 Vmw175 DNA binding domain with its varicella-zoster virus counterpart results in a protein with novel regulatory properties that can support virus growth.

The alphaherpesviruses encode major immediate early transactivator proteins that are essential for the expression of later classes of viral genes. We have previously shown that the extensive sequence similarity between the herpes simplex virus type 1 (HSV-1) and varicella-zoster virus (VZV) members of the family (proteins Vmw175 and VZV140k) extends to function, since a virus which expresses VZV140k in place of Vmw175 is able to grow, albeit at reduced efficiency. We have also shown that the DNA binding characteristics of the isolated DNA binding domains of Vmw175 and VZV140k are related but distinct. In order to assess whether the different DNA binding properties of the two proteins are responsible for the differences in their individual transcriptional regulatory functions, we constructed a plasmid and an HSV-1 virus in which the VZV140k DNA binding domain coding sequences replace those of Vmw175. The characteristics of the resultant hybrid protein in transfection assays and during virus infection suggest that the nature of the DNA binding domain plays a significant role in the transactivation and repression properties of the Vmw1 75 family of proteins.

Animals↗
PubMed1997

Depression after childbirth: the views of medical students and women compared.

BACKGROUND: Little research has been conducted on the views of health professionals about women's experiences of depression after childbirth. This study compared the views held by undergraduate medical students about postnatal depression with those of women who had themselves experienced it. METHODS: Fourth- and sixth-year medical students at one Australian university were surveyed (n = 134). Their views about prevalence, duration, contributing factors, and advice for dealing with postnatal depression were compared with the findings from 60 women in a population-based study of mothers who gave birth in Victoria in 1989, in which women scoring as depressed 8 to 9 months after birth on the Edinburgh Postnatal Depression Scale were interviewed 12 to 18 months later about their experiences of depression and their advice to other mothers. RESULTS: Women's and students' views differed markedly, with students much more likely to view hormonal and biologic factors and a "tendency to depression" as playing an important role than women who identified a wide range of social, physical health, and life event factors as contributing to their experience of depression. Fourth-year students tended to overestimate the prevalence of depression and sixth-year students to underestimate it. Both groups underestimated the duration of depression compared with women's actual experiences. CONCLUSION: Medical students need to develop a broader understanding of maternal depression after the birth of a baby, and women's own views of the experience can and should make an important contribution to medical teaching on this topic.

Attitude of Health Personnel↗
PubMed1995

Separation of sequence requirements for HSV-1 Vmw110 multimerisation and interaction with a 135-kDa cellular protein.

Herpes simplex virus type 1 immediate-early polypeptide Vmw110 (ICP0) is a general transactivator of gene expression in transfection assays and is required for the fully efficient onset of viral lytic replication. It has also been implicated in the process of viral reactivation from latency. Its mechanism of action is unknown, but any involvement in latency requires interactions between viral and host factors. We have previously shown that Vmw110 binds to a 135-kDa cellular protein. In this paper we define a short region towards the C-terminal end of Vmw110 that is required for the 135-kDa protein interaction in virus-infected cells and in vitro. We also confirm that the C-terminal region of Vmw110 contains residues that are responsible for the multimerisation of the protein; these sequences are at least partially distinct from those involved in 135-kDa binding. Both multimerisation and 135-kDa protein interaction are required for full viral infectivity, and elimination of these functions affects the normal interactions between Vmw110 and cellular nuclear structures that contain the PML protein.

Animals↗
PubMed1995

The cellular RING finger protein PML is not a functional counterpart of the herpes simplex virus type 1 RING finger protein Vmw110.

Herpes simplex virus type 1 (HSV-1) immediate early protein Vmw110 (also known as ICP0) is required for the fully efficient expression of viral genes during onset of lytic growth and for normal reactivation from latency. Both Vmw110 and the cellular protein PML are members of the RING finger family of zinc binding domain proteins, a family which includes an increasing number of examples from a wide evolutionary range. The function of the RING finger domain is unknown, and the question arises whether the RING finger (like several other examples of conserved domains) fulfils similar functions in these diverse proteins. Another link between Vmw110 and PML is that at early times of HSV-1 infection Vmw110 migrates to distinct nuclear structures which contain the PML protein. In order to test the possibility that PML and Vmw110, or their RING finger domains, fulfill similar functions, we have constructed recombinant viruses that express either intact PML, or a chimeric Vmw110 protein which contains the PML RING finger in place of its own. The results indicate that the PML and Vmw110 RING fingers are not functionally interchangeable, and that PML is not a cellular functional counterpart of Vmw110.

Animals↗
PubMed1995

The equine herpesvirus 1 gene 63 RING finger protein partially complements Vmw110, its herpes simplex virus type 1 counterpart.

All alpha herpesviruses of known DNA sequence have been found to encode a protein with similarities to immediate early protein Vmw110 (ICP0) of herpes simplex virus type 1 (HSV-1). The conserved portion of this family of proteins is a characteristic zinc binding module, known as a RING finger or C3HC4 domain. Examples of RING finger domains occur in many other proteins of diverse evolutionary origin and function. Recently, the solution structure of the equine herpesvirus 1 (EHV-1) RING finger protein, encoded by gene 63, has been solved. To investigate whether this structure could be considered to be a paradigm of herpesvirus RING domains, we have constructed a recombinant HSV-1 which expresses the EHV-1 gene 63 protein (EHVg63) in place of Vmw110. Comparison of the growth properties of the recombinant with those of wild-type and Vmw110-defective viruses indicates that EHVg63 is able to fulfil partially, but not completely, the roles of Vmw110 during virus growth in tissue culture.

Animals↗
PubMed1995

Point mutations in the herpes simplex virus type 1 Vmw110 RING finger helix affect activation of gene expression, viral growth, and interaction with PML-containing nuclear structures.

Herpes simplex virus type 1 immediate-early protein Vmw110 (also known as ICP0) has been implicated in the control of the balance between the lytic and latent states, but the precise mechanisms by which it exerts its effects are unknown. Vmw110 includes a characteristic zinc binding domain, termed the C3HC4 domain or RING finger, which is essential for its function. The solution structure of a related herpesvirus RING finger domain suggested that an amphipathic alpha helix might be an important functional component of the RING finger. In this paper, we show that the equivalent region of Vmw110 is important for virus growth in tissue culture and for the normal interaction of Vmw110 with nuclear structures which include the PML protein.

Amino Acid Sequence↗
PubMed1994

Herpes simplex virus type 1 immediate-early protein Vmw110 binds strongly and specifically to a 135-kDa cellular protein.

Herpes simplex virus type 1 encodes five immediate-early gene products, at least three of which are required for fully efficient viral gene expression. One of these three, Vmw110 (or ICPO), is a potent and nonspecific activator of gene expression in transfection assays. Viruses which fail to express functional Vmw110 have a cell-type and multiplicity-dependent defect in viral gene expression. In addition, Vmw110 has been implicated in the reactivation of latent virus in mouse and tissue culture model systems. In this report we show that Vmw110 can be isolated as a fast-sedimenting complex from infected cells and that this complex contains a cellular protein which is immune precipitated in association with Vmw110. This association can be reconstructed by adding purified Vmw110 to lysates of several different cell types. By using a GST fusion protein, we have found that the isolated C-terminal portion of Vmw110 can complex strongly and specifically with a similar cellular protein. The relevance of this observation to the roles of Vmw110 in latent and lytic virus infection is discussed.

Animals↗
PubMed1994

Synthesis and DNA-sequence selectivity of a series of mono- and difunctional 9-aminoacridine nitrogen mustards.

The aim of this work was to identify nitrogen mustards that would react selectively with DNA, particularly in G-rich regions. A series of mono- and difunctional nitrogen mustards was synthesized in which the (2-chloroethyl)amino functions were connected to the N9 of 9-aminoacridine by way of a spacer chain consisting of two to six methylene units. The length of the spacer chain connecting the alkylating and putative DNA-intercalating groups was found to affect the preference for the alkylation of different guanine-N7 positions in a DNA sequence. All of the compounds reacted preferentially at G's that are followed by G as do most other types of nitrogen mustards, but the degree of selectivity was greater. The compounds reacted at much lower concentrations than were required for comparable reaction by mechlorethamine (HN2), consistent with initial noncovalent binding to DNA prior to guanine-N7 alkylation. The degree of DNA-sequence selectivity increased as the spacer-chain length decreased below four methylene units. Most strikingly, long spacer compounds reacted strongly at 5'-GT-3' sequences, whereas this reaction was almost completely suppressed when the spacer length was reduced to two or three methylenes. Mono- and difunctional compounds of a given spacer length showed no consistent difference in DNA-sequence preference.

Acridines↗
PubMed1994

The relative expression of human histone H2A genes is similar in different types of proliferating cells.

To help elucidate the factors regulating the expression of histone multigene families in proliferating cells, we asked whether the relative expression of different members of such a family was dependent upon or independent of the type of proliferating cell. This question was examined by measuring the relative expression of seven members of the human histone H2A multigene family in four cell lines of diverse origin. Two previously uncharacterized members of the H2A gene family were found to be the most abundantly expressed of the seven in all four cell lines. One of these encodes an H2A.2 species containing methionine. The lines examined in the study were Jurkat (a lymphoma line), N-tera (a pluripotent embryonic carcinoma line), HeLa (originally isolated as a cervical carcinoma), and IMR90 (a normal embryonic fibroblastic line). The amount of each mRNA species was quantitated using oligonucleotides about 30 bases long complementary to the 5' or 3' untranslated regions. In each cell line, there was at least an eight-fold difference in the amount of the most and least highly expressed of the seven H2A mRNA species. In addition, there were up to five-fold differences among the cell lines in the amount of the H2A mRNA species as a fraction of total RNA. However, in contrast to those differences, the four cell lines were found to express the seven H2A mRNAs in similar relative amounts. These findings suggest that the relative expression of the individual members of a histone gene family is independent of the type of replicating cell.

Amino Acid Sequence↗
PubMed1993

A novel arrangement of zinc-binding residues and secondary structure in the C3HC4 motif of an alpha herpes virus protein family.

A highly conserved, cysteine-rich region plays a crucial role in the function of a family of regulatory proteins encoded by alpha herpes viruses. The so-called C3HC4 motif spans approximately 60 residues and has been predicted to bind zinc. This motif occurs in a number of other viral and cellular proteins, many of which appear to be involved in some aspect of the regulation of gene expression. We have cloned and expressed in bacteria a portion of immediate-early protein Vmw110 of herpes simplex virus type 1 that encompasses the C3HC4 motif, and the equivalent regions from the homologous proteins of varicella zoster virus and equine herpes virus type 1 (EHV-1). All three polypeptides were purified and found to bind zinc stably. None of the three interacted significantly with either DNA or RNA under our assay conditions. The EHV-1 domain yielded interpretable proton nuclear magnetic resonance spectra. Assignment of resonances and analysis of nuclear Overhauser effects revealed its secondary structure. Starting from the N terminus, this consists of an ordered but irregular loop, the first two strands of a triple-stranded antiparallel beta-sheet, two turns of an alpha-helix, a second irregular loop, and the third strand of the beta-sheet. It appears that, taking the cysteine and histidine residues in turn, cysteine residues I, II, IV and V co-ordinate one zinc atom while the histidine residue and cysteine residues III, VI and VII co-ordinate a second zinc atom. This arrangement of secondary structure differs from that found in other characterized zinc-containing proteins.

Amino Acid Sequence↗
PubMed1993

A truncated form of herpes simplex virus type 1 immediate-early protein Vmw110 is expressed in a cell type dependent manner.

Herpes simplex virus type 1 (HSV-1) encodes five immediate-early (IE) genes, at least three of which are involved in the regulation of gene expression. Gene IE-1 is one of the few HSV-1 genes whose pre-mRNAs are spliced; the IE-1 pre-mRNA contains two introns, the second of which contains an in-frame stop codon which would terminate IE-1 translation if the intron were not excised. Previous work has shown that plasmids which have been constructed so as to express only the first two exons of Vmw110 can inhibit gene expression in transfection assays, whereas the normal intact protein is an activator of gene expression. In this paper we show that this predicted truncated Vmw110 protein is expressed during normal HSV-1 infection, and that it must be translated from IE-1 pre-mRNAs which retain the in-frame stop codon in the second intron. This truncated product is produced in amounts which depend upon the cell type infected. The possible consequences of these observations are discussed.

Animals↗
PubMed1993

An epitope within the DNA-binding domain of the herpes simplex virus immediate early protein Vmw175 is conserved in the varicella-zoster virus gene 62 protein.

We have isolated a panel of monoclonal antibodies that recognize the DNA-binding domain of the herpes simplex virus type 1 (HSV-1) immediate early polypeptide Vmw175. The mice used for the fusions had been immunized with the isolated Vmw175 DNA-binding domain. This had been purified from bacteria that carried a phage T7 expression plasmid with the DNA-binding domain coding region. The epitopes recognized by the monoclonal antibodies were mapped by using a family of truncated versions of the DNA-binding domain, which had also been expressed in the bacterial expression system. The monoclonal antibodies divided into at least four different groups according to this mapping. Several of the monoclonal antibodies recognized Vmw175 expressed in infected BHK cells by HSV-1 strain 17 in Western blots. One of them also recognized the corresponding protein of varicella-zoster virus gene 62. This is further illustration of the relatedness of the two polypeptides.

Amino Acid Sequence↗
PubMed1993

Histone H2A.X gene transcription is regulated differently than transcription of other replication-linked histone genes.

Histone H2A.X is a replication-independent histone H2A isoprotein species that is encoded by a transcript alternatively processed at the 3' end to yield two mRNAs: a 0.6-kb mRNA ending with the stem-loop structure characteristic of the mRNAs for replication-linked histone species, and a second, polyadenylated 1.6-kb mRNA ending about 1 kb further downstream (C. Mannironi, W. M. Bonner, and C. L. Hatch, Nucleic Acids Res. 17:9113-9126, 1989). Of the two, the 0.6-kb H2A.X stem-loop mRNA predominates in many cell lines, indicating that the presence of two types of mRNA may not completely account for the replication independence of H2A.X protein synthesis. The ambiguity is resolved by the finding that the level of the 0.6-kb H2A.X mRNA is only weakly downregulated during the inhibition of DNA replication and only weakly upregulated during the inhibition of protein synthesis, while the levels of other replication-linked mRNAs are strongly down- or upregulated under these two conditions. Analysis of the nuclear transcription rates of specific H2A genes showed that while the rates of transcription of replication-linked H2A genes decreased substantially during the inhibition of DNA synthesis and increased substantially during the inhibition of protein synthesis, the rate of H2A.X gene transcription decreased slightly under both conditions. These differences in transcriptional regulation between the H2A.X gene and other replication-linked histone genes are sufficient to account for the differences in regulation of their respective stem-loop mRNAs.

Base Sequence↗
PubMed1992

Rational design and molecular effects of a new topoisomerase II inhibitor, azatoxin.

Azatoxin [NSC 640737-M; 5.R,11aS-1H,6H,3-one-5,4,11,11a-tetrahydro-5-(3,5-dimethoxy-4-hydr oxyphenyl) oxazolo (3',4':1,6)pyrido-(3,4-b)indole] was rationally designed from a model for the pharmacophore of drugs with topoisomerase II inhibition activity. This pharmacophore has at least 2 domains: a quasiplanar polycyclic ring system proposed to bind between the DNA base pairs and a pendant substituent proposed to interact with the enzyme and/or to the DNA grooves. The present study shows that, in cell free systems, azatoxin induces a large number of double strand-breaks in linear Simian virus 40 and human c-myc DNA. These breaks yield cleavage patterns that are different from those of well established topoisomerase II inhibitors (epipodophyllotoxins, amsacrine, mitoxantrone). Azatoxin also inhibits the catalytic activity of purified topoisomerase II, and is a nonintercalator. The structure-activity relationship of 3 isomers and 6 derivatives of azatoxin shows a stringent stereochemical requirement for activity. The effects of azatoxin pendant ring substitution on topoisomerase II mediated DNA cleavage activity were similar to the relationship observed for etoposide.

DNA↗
PubMed1992

Differential effects of amsacrine and epipodophyllotoxins on topoisomerase II cleavage in the human c-myc protooncogene.

Amsacrine and demethylepipodophyllotoxins (etoposide and teniposide) are potent topoisomerase II inhibitors which have optimum activity in different cancers. To investigate whether these differences are due to different activity on cellular oncogenes, drug-induced topoisomerase II cleavage sites were mapped and sequenced in the human c-myc protooncogene. In the presence of purified murine L1210 topoisomerase II, amsacrine induces prominent cleavage in the P2 promoter (site 2499/2502). Footprinting experiments indicate that topoisomerase II binds to the entire promoter region (approximately 20 base pairs on the sides of the P2 site). In the case of teniposide or etoposide, cleavage is more diffuse and markedly less at the P2 site. Mapping of cleavage sites in human small cell lung carcinoma cells (NCI N417) also shows that cleavage in the P2 promoter region is induced preferentially by amsacrine but not by demethylepipodophyllotoxins. Thus, selective gene damage among topoisomerase II inhibitors may contribute to differential anticancer activity.

Amsacrine↗
PubMed1991

Distribution of topoisomerase II cleavage sites in simian virus 40 DNA and the effects of drugs.

The distributions of DNA cleavage sites induced by topoisomerase II in the presence or absence of specific drugs were mapped in the simian virus 40 genome. The drugs studied were 5-iminodaunorubicin, amsacrine (m-AMSA), teniposide (VM-26) and 2-methyl-9-hydroxyellipticinium; each produced a distinctive pattern of enhanced cleavage. Consistently intense cleavage, both in the presence and in the absence of drugs, occurred in the nuclear matrix-associated region. Since topoisomerase II is a major constituent of the nuclear matrix, and cleavage complexes include a covalent link between topoisomerase II and DNA, the findings suggest that topoisomerase II may function to attach DNA to the nuclear matrix. Cleavage usually occurred on both DNA strands with the expected four base-pair 5' stagger, and strong sites tended to occur within A/T runs such as have been associated with binding to the nuclear scaffold. Intense cleavage was present also in the replication termination region, but was absent from the vicinity of the replication origin. Cleavage intensities were found to change with time in a manner that depended both on the site and on the drug, suggesting that topoisomerase II can move along the DNA from a kinetically preferred site to a thermodynamically preferred site.

Amsacrine↗