Human cytomegalovirus immediate early interaction with host nuclear structures: definition of an immediate transcript environment.

The development of an induced transcript environment was investigated at the supramolecular level through comparative localization of the human cytomegalovirus immediate early (IE) transcripts and specific nuclear domains shortly after infection. Compact aggregates of IE transcripts form only adjacent to nuclear domain 10 (ND10), and the viral protein IE86 accumulates exclusively juxtaposed to the subpopulation of ND10 with transcripts. The stream of transcripts is funneled from ND10 into the spliceosome assembly factor SC35 domain through the accumulation of IE86 protein, which recruits some components of the basal transcription machinery. Concomitantly the IE72 protein binds to ND10 and later disperses them. The domain containing the zinc finger region of IE72 is essential for this dispersal. Positional analysis of proteins IE86 and IE72, IE transcripts, ND10, the spliceosome assembly factor SC35, and basal transcription factors defines spatially and temporally an immediate transcript environment, the basic components of which exist in the cell before viral infection, providing the structural environment for the virus to usurp.

Fibroblasts and macrophages of mice with the Chediak-Higashi-like syndrome have microtubules and actin cables.

Cells of the beige mouse contain abnormally large lysosomes and show enhanced capping of concanavalin A. It has been suggested that these phenomena may be secondary to a defect in microtubule polymerization. We have examined the cytoskeleton of beige mouse cells by indirect immunofluorescence and find the number and distribution of microtubules and actin cables to be indistinguishable from those of normal control cells.

Characterization of a phosphoprotein associated with the SS-B/La nuclear antigen in adenovirus-infected and uninfected KB cells.

We have employed sera from patients with autoimmune disease to characterize the nuclear SS-B/La antigen in uninfected and adenovirus-infected KB cells. A 45,000-dalton phosphorylated polypeptide was specifically precipitated with anti-SS-B sera, and the level of phosphorylation was increased after infection. The increased phosphorylation appears to occur at the same amino acid residues phosphorylated in uninfected cells and results from increased phosphorylating activity rather than from altered enzyme specificity. A competition experiment between infected and uninfected cell extracts indicates that the antigen in the infected cell binds more strongly to SS-B/La antibodies. Fragments of adenovirus-induced virus-associated RNA as well as intact molecules complex with SS-B/La antigen and are immune precipitated with autoimmune sera.

Simultaneous measurement of coronary flow reserve by left anterior descending coronary artery Doppler and great cardiac vein thermodilution methods.

OBJECTIVES: The objective of this study was to compare left anterior descending coronary artery Doppler blood flow velocity and great cardiac vein thermodilution blood flow measurements of coronary flow reserve and submaximal coronary vasodilation in humans. BACKGROUND: Reported maximal coronary flow reserve values obtained with the coronary venous thermodilution method are lower than those obtained with other measurement methods. METHODS: Thermodilution measurements of great cardiac vein flow in 11 subjects were compared with simultaneous Doppler measurements of changes in left anterior descending coronary flow velocity after intracoronary administration of papaverine, nitroglycerin, iohexol and intravenous administration of dipyridamole. RESULTS: Coronary flow reserve (papaverine peak/rest flow ratio) was 3.7 +/- 1.7 (mean +/- SD) by the Doppler method and 2.0 +/- 0.7 by the thermodilution technique (p less than 0.001). Thermodilution flow changes were also smaller than Doppler-measured changes during submaximal vasodilation and during prolonged coronary dilation after dipyridamole administration. CONCLUSIONS: Coronary flow reserve and submaximal flow increases measured with the thermodilution method were consistently and substantially smaller than Doppler-derived measurements. This discrepancy has important implications for the comparison of coronary flow reserve measurements performed with the use of different techniques.

Comparison of coronary vasodilation with intravenous dipyridamole and adenosine.

Although both intravenous dipyridamole and adenosine have been used to produce coronary vasodilation during cardiac imaging, the relative potency of the commonly administered doses of these agents has not been evaluated. Accordingly, the coronary and systemic hemodynamic effects of intravenous adenosine (140 micrograms/kg per min) and intravenous dipyridamole (0.56 mg/kg over 4 min) were compared with a maximally dilating dose of intracoronary papaverine in 15 patients. Coronary blood flow responses were assessed using a Doppler catheter in a nonstenotic coronary artery. The protocol was discontinued in two patients because of transient asymptomatic atrioventricular (AV) block during adenosine infusion. The mean heart rate increased more with adenosine (11 +/- 9 beats/min) and dipyridamole (11 +/- 7 beats/min) than with papaverine (4 +/- 3 beats/min, p less than 0.05 vs. adenosine and papaverine). The mean arterial pressure decreased less with dipyridamole (-10 +/- 3 mm Hg) and papaverine (-9 +/- 4 mm Hg) than with adenosine (-16 +/- 5 mm Hg, p less than 0.01 vs. dipyridamole and papaverine). The peak/rest coronary blood flow velocity ratio was greater with papaverine (3.9 +/- 1.1) than with adenosine (3.4 +/- 1.2, p less than or equal to 0.05 vs. papaverine) or dipyridamole (3.1 +/- 1.2, p less than 0.01 vs. papaverine). A larger decrease in coronary resistance as measured by the coronary vascular resistance index occurred with papaverine (0.25 +/- 0.06) and adenosine (0.26 +/- 0.09) than with dipyridamole (0.31 +/- 0.10, p less than 0.01 vs. papaverine, p less than 0.05 vs. adenosine).(ABSTRACT TRUNCATED AT 250 WORDS)

Left ventricular dysfunction due to chronic right ventricular pressure overload. Resolution following percutaneous balloon valvuloplasty for pulmonic stenosis.

Left ventricular dysfunction due to chronic right ventricular pressure overload is well documented in experimental animals, but is controversial in humans. Whether left ventricular dysfunction resolves following the relief of chronic right ventricular pressure overload has not been studied. In this report, rapid improvement in both right and left ventricular function following successful percutaneous balloon valvuloplasty is described in a patient with severe isolated valvular pulmonic stenosis and biventricular dysfunction. It appears that: (1) geometric distortion played a major role in his reversible left ventricular dysfunction, and (2) severe biventricular dysfunction should not be a contraindication to valvuloplasty for valvular pulmonic stenosis.

Simultaneous acute thrombosis of two major coronary arteries following intravenous cocaine use.

Myocardial infarction is now a well-recognized complication of cocaine abuse. This report describes a 38-year-old man who sustained simultaneous acute thrombosis of two major epicardial coronary arteries shortly after intravenous cocaine use. The finding of layers of mural thrombus of varying age, from acute to two to three days, in both coronary arteries represents a previously unreported finding (to our knowledge) in cocaine-associated cardiac death. Potential mechanisms for the association between cocaine use and infarction and the cardiac pathologic findings in cocaine-associated death are discussed.

Emergency coronary stenting for complete thrombotic occlusion of an unprotected left main coronary artery in acute myocardial infarction complicated by cardiogenic shock in an octogenarian patient--a case report.

This report concerns an 82-year-old white man, who was admitted with cardiogenic shock secondary to an acute anterior myocardial infarction with right bundle branch block requiring an intra-aortic balloon pump for hemodynamic support and mechanical ventilatory support for respiratory distress. An immediate cardiac catheterization with coronary angiography revealed a complete thrombotic occlusion of the left main coronary artery. Prompt stent-supported percutaneous transluminal coronary angioplasty to the occluded left main coronary artery, a critical stenosis of the ostial left anterior descending artery, and the left circumflex coronary artery, allowed for recovery from this life-threatening condition and subsequent discharge from the hospital of this octogenarian patient. It is suggested that in a critical clinical condition with particularly challenging coronary anatomical findings, stent-supported coronary angioplasty can be lifesaving treatment in selected patients with octogenarian status with acute myocardial infarction.

Is there a connection between severe cerebral palsy and increased gluten sensitivity?

UNLABELLED: In nine children with severe cerebral palsy (CP), feeding difficulties and poor development of weight and height, laboratory markers for metabolic and enteral dysfunction were studied. CONCLUSION: Four of the nine patients with CP had increased levels of antigliadin antibodies AGA (IgA), a finding which calls for further studies concerning the possible connection between increased celiac markers and CP.

The human cytomegalovirus major immediate-early gene.

Immediate-early genes function to regulate viral and cellular gene expression during the course of virus replication. The major immediate-early gene region of human cytomegalovirus plays a key role in affecting activation and repression of viral and cellular genes. These proteins intimately associate and/or interact with viral and cellular proteins during this process. Herein, we will discuss the current understanding of this complex gene region.

Clinical evaluation of glass ceramic inlays (Dicor).

The purpose of the study was to evaluate the clinical behavior of ceramic class-II inlays (Dicor) in the first 2 years after placement. As a reference, a similar number of dental amalgam restorations were followed up during the same period. Twenty-five inlays and 25 dental amalgams were placed on premolars and first molars of 20 and 19 patients (15-19 years old), respectively. The inlay preparations were made in accordance with the manufacturer's recommendations, and the inlays were produced by a licensed Dicor laboratory. The inlays were luted, using a glass ionomer cement. The dental amalgam preparations were made using standard class-II preparation techniques and filled with ANA 2000. The inlays were evaluated after 6, 12, and 24 months, and the dental amalgam restorations after 24 months, using the criteria suggested by Ryge. In addition, the 24-month examination included proximal recording of dental plaque and gingivitis. With the exception of two inlays that fractured during the observation period, all ceramic inlays showed excellent ratings for anatomic form, marginal discoloration, and marginal caries at all examinations. Two inlays showed minor marginal defects but were classified within the range of acceptance with no need for replacement. The two fractured inlays were replacements of earlier fractured dental amalgams. The clinical behavior of the dental amalgam restorations was in most respects similar to that of the ceramic inlays. Unlike the inlays, however, no dental amalgams fractured during the observation period.(ABSTRACT TRUNCATED AT 250 WORDS)

Regulated expression of the human cytomegalovirus pp65 gene: octamer sequence in the promoter is required for activation by viral gene products.

To better understand the regulation of late gene expression in human cytomegalovirus (CMV)-infected cells, we examined expression of the gene that codes for the 65-kilodalton lower-matrix phosphoprotein (pp65). Analysis of RNA isolated at 72 h from cells infected with CMV Towne or ts66, a DNA-negative temperature-sensitive mutant, supported the fact that pp65 is expressed at low levels prior to viral DNA replication but maximally expressed after the initiation of viral DNA replication. To investigate promoter activation in a transient expression assay, the pp65 promoter was cloned into the indicator plasmid containing the gene for chloramphenicol acetyltransferase (CAT). Transfection of the promoter-CAT construct and subsequent superinfection with CMV resulted in activation of the promoter at early times after infection. Cotransfection with plasmids capable of expressing immediate-early (IE) proteins demonstrated that the promoter was activated by IE proteins and that both IE regions 1 and 2 were necessary. Analysis of promoter deletion mutants indicated that the 5' minimal sequence required for activation is -61 from the CAP site (+1) and that an 8-base-pair sequence located at -51 to -58 is necessary for activation of the pp65 promoter. This sequence is repeated once at +93 and is found as an inverted repeat at +67. These studies suggest that interactions between IE proteins and this octamer sequence may be important for the regulation and expression of this CMV gene.

Functional analysis of the true late human cytomegalovirus pp28 upstream promoter: cis-acting elements and viral trans-acting proteins necessary for promoter activation.

As a model for analyzing the regulation of human cytomegalovirus late genes, we investigated the 28-kDa phosphoprotein (pp28) gene region. Transcripts of 1.6 and 1.3 kb were expressed in wild-type human cytomegalovirus-infected cells but not in cells infected with a DNA-negative temperature-sensitive mutant (ts66), indicating that DNA replication is absolutely required for pp28 gene expression. Transient promoter activation studies revealed that the pp28 gene region upstream promoter (pp28US) functioned early when expressed independently of the viral genome. However, the promoter was not efficiently activated by immediate-early (IE) proteins but was activated equally well by both wild-type virus and ts66. Deletion analysis of the pp28US promoter indicated that sequences upstream of the CAP site between -107 and -32 were required for activation of the pp28 promoter. Within that region exist a 10-bp sequence at -90 (AGTGAT CGTG) and its inverted repeat at -32 which positively influence pp28 promoter function. Therefore, in the case of the pp28US promoter, viral proteins interact through discrete sequences to facilitate late gene expression.

Cytomegalovirus genes: their structure and function.

During the past several years, studies have indicated that cytomegalovirus (CMV) genes can be grouped into two broad categories; those essential for replication in cell culture and those dispensible for virus replication. The latter group of genes are likely to be important for pathogenesis and host-virus interactions. As the field progresses, the need to utilize and establish biological systems capable of addressing gene function during a natural infection of cells in culture, or in the infected animal, is becoming more apparent. Herein, we describe the current status of some of those systems, what has been learned and where these studies may lead. Specifically, we address studies that assess mechanisms of gene activation and function in biologically relevant systems. These include (i) the identification of genes dispensible for replication in cell culture, (ii) the use of dispensible regions of the CMV genome to manipulate genetic information for assessing gene function and activation, and (iii) the identification of a related group of essential loci important for replication of human CMV (HCMV) DNA and what is presently known of the function of those genes during HCMV infection.

Modulation of herpes simplex virus replication in adenovirus transformed cells.

The ability of herpes simplex virus 1 to replicate in cells transformed by adenovirus type 5 is strongly dependent on the origin of the cells. Studies show that adenovirus transformed rat cells lose their permissiveness while cells of hamster or human origin retain their ability to replicate HSV although at a reduced level when compared to the untransformed parent cells. One line of adenovirus transformed rat cells, 107, demonstrates thermosensitive events, allowing HSV to replicate at 34 degrees C but not at 37 degrees C. Analysis of the biochemical events taking place at 37 degrees C showed that virus-specific DNA synthesis was greatly reduced but that all of the late virus structural proteins could be observed after SDS-polyacrylamide gel electrophoresis. It was also demonstrated that shut-off of host macromolecular synthesis appeared to be less efficient after HSV infection of 107 cells than after infection of more permissive cells such as the non-transformed REF line. Collectively the data show that interactions between HSV and the host cell are perturbed when the cell is transformed by type 5 adenovirus. The degree of perturbation ranges from a slight reduction in number of progeny to a completely abortive infection.

Herpes simplex virus-induced changes in cellular and adenovirus RNA metabolism in an adenovirus type 5-transformed human cell line.

We used the viral transcripts (designated Ad-RNA) that accumulated in the cytoplasm of adenovirus type 5-transformed human embryonic kidney cells (cell line 291-31) as models for cellular RNAs to examine how herpes simplex virus modifies cellular RNA metabolism. Infection of 293-31 cells with herpes simplex virus type 1 strain 17 lead to extensive inhibition of Ad-RNA accumulation by 4 h postinfection. The major part of this inhibition was due to an immediate early or alpha gene function, which reduced the rate of transcription of Ad-RNA within the nuclei of the infected cells. In addition, host polyadenylic acid-containing RNA accumulation and rRNA accumulation were affected, but to a lesser extent and at lower rate than Ad-RNA accumulation. In conjunction with previous data, our experimental data allowed us to propose a general scheme for how herpes simplex virus type 1 alters the metabolism of cellular RNA, the possible mechanisms for these changes, and how they correlate with the regulation of herpes simplex virus gene expression.

Autoregulation of the human cytomegalovirus major immediate-early gene.

The gene coding for the human cytomegalovirus major immediate-early 72-kilodalton protein was cloned into simian virus origin of DNA replication plasmid pSVOd. Transfection of this plasmid (pSVCC2) into cells constitutively expressing the simian virus 40 T-antigen resulted in readily detectable levels of immediate-early region 1-specific RNA and protein. Partial restriction enzyme digestion of pSVCC2 was used to generate specific amino acid deletions within the 72-kilodalton protein. Mutant delta S12, which contained a deletion of 145 amino acids at the carboxy terminus of the protein, accumulated at least 10 times more immediate-early region 1 RNA than wild-type pSVCC2 did. In contrast, normal levels of delta S12-specific RNA were detected in cells cotransfected with wild-type pSVCC2. Therefore, the wild-type gene was capable of suppressing transcription of the mutant gene. Our results suggest that the wild-type major immediate-early protein of cytomegalovirus autoregulates transcription of immediate-early region 1 and that one of the regulatory domains is within the carboxy-terminal 145 amino acids of the viral protein.

Durability of tunnel restorations in general practice: a three-year multicenter study.

Twelve dentists, clinically experienced and familiar with the tunnel technique, placed 374 tunnel restorations in permanent teeth. Mean age of the patients was 19.1 years (range 10-74). The filling material used was a glass cermet cement, Ketac Silver. After 1, 2 and 3 years the teeth were controlled by the dentists. The bitewing radiographs from baseline, 1, 2 and 3 years were also analyzed by 2 of the authors, independently. The baseline radiographs showed technical defects in 6% and indicated remaining dentin caries in 8% of the restorations. After 3 years, 305 restorations were accessible for examination. The cumulative replacement rate was 20%. The main reasons for replacement were marginal ridge fracture (14%) and dentin caries (3%). The number of restorations showing untreated progressive caries increased during the study. After 3 years, untreated dentin caries was seen in 28 cases (11%) and almost half of the left enamel lesions showed progression.

Structural analysis of the major immediate early gene of human cytomegalovirus.

The most abundant species of human cytomegalovirus (Towne) immediate early polysome-associated RNA originates from a region of ca. 2.8 kilobases (0.739 to 0.755 map units) within the XbaI-E DNA fragment. These sequences code for a 1.95-kilobase mRNA and are referred to as immediate early coding region one (M. F. Stinski, D. R. Thomsen, R. M. Stenberg, and L. C. Goldstein, J. Virol. 46:1-14, 1983). We have utilized the nuclease mapping technique of Berk and Sharp (A. J. Berk and P. A. Sharp, Cell 12:721-732, 1977) to examine this gene in detail. Cloned fragments of human cytomegalovirus DNA, either labeled with 32P in vivo or end labeled in vitro at the 5' or 3' termini, were hybridized to immediate early polysome-associated RNA. The hybrids were treated with single-strand-specific nuclease and subjected to electrophoresis in either neutral or denaturing gels. The major transcript was shown to be a spliced molecule containing a 3' terminal exon of 1,341 nucleotides. Upstream of the major body of the mRNA are three small exon sequences of 185, 88, and 121 nucleotides. The sequence of the exons as well as the locations of the intron-exon splice junctions were determined. Based on the DNA sequence, the viral mRNA molecule has one open reading frame which begins within the second exon and extends for 491 amino acid residues. The predicted molecular weight of the polypeptide originating from this region was estimated to be 64,000. It is hypothesized that this viral gene codes for the major regulatory protein controlling transcription of the viral genome at early times. The properties of the viral gene and its protein product are discussed.

Identification of sequence elements in the human cytomegalovirus DNA polymerase gene promoter required for activation by viral gene products.

To determine the mechanisms involved in the regulation of human cytomegalovirus early gene expression, we have examined the gene that encodes the viral DNA polymerase (UL54, pol). Our previous studies demonstrated that sequences required for activation of the pol promoter by immediate-early proteins are contained within a region from -128 to +20 and that cellular proteins can bind to this activation domain. In this study, we demonstrate by competition analysis that binding of cellular proteins to pol is associated with an 18-bp region containing a single copy of a novel inverted repeat, IR1. Time course analysis indicated that viral infection increased the level of protein binding to IR1, concurrent with the activation of the pol promoter. Mutation of the IR1 element abrogated binding of cellular factors to the pol promoter and reduced by threefold the activation by immediate-early proteins. Similarly, mutation of IR1 rendered the promoter poorly responsive to activation by viral infection. Mutation of additional sequence elements in the pol promoter had little effect, indicating that IR1 plays the major role in pol promoter regulation. These studies demonstrate that the interaction between cellular factors and IR1 is important for the regulation of expression of the polymerase gene by viral proteins.