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Biomedical subjects

J A Rose

Publications and source records attributed to J A Rose.

At least 19 recordsLinked to original sources
PubMed1995

Significance of peritoneal cytology in patients with potentially resectable adenocarcinoma of the pancreatic head.

BACKGROUND: Recurrence in the peritoneum occurs in up to 50% of patients after a potentially curative pancreaticoduodenectomy. Previous authors have implicated preoperative fine-needle aspiration (FNA) as a cause of intraperitoneal tumor dissemination, although prior studies of peritoneal cytology findings have largely involved patients with locally advanced disease. METHODS: A consecutive series of patients referred to our institution between 1991 and 1993 with suspected or biopsy-proven adenocarcinoma of the pancreatic head was studied prospectively. All patients fulfilled criteria for resectability as assessed by computed tomography: no metastatic disease, no encasement of the superior mesenteric or hepatic arteries, and a patent superior mesenteric-portal venous confluence. Peritoneal washings were obtained at the time of staging laparoscopy and/or at subsequent laparotomy. Data regarding peritoneal cytology results, previous FNA, preoperative chemoradiation, eventual resection, pattern of disease recurrence, and survival were collected. RESULTS: A total of 80 peritoneal washings from 60 consecutive patients were prospectively examined. Forty-nine (82%) of 60 patients underwent FNA before peritoneal washings were obtained. A total of four patients (7%) had positive peritoneal cytology findings: three (6%) of 49 who underwent prior FNA and one (9%) of 11 with no prior FNA. Similarly, no differences in eventual peritoneal failure or short-term survival were observed for patients who underwent prior FNA compared with patients who did not. All four patients with positive peritoneal cytology findings had metastatic disease (liver, three; peritoneum, one) at a median of 4.8 months after diagnosis; three of the four died of disease at a median of 8 months. CONCLUSIONS: Positive peritoneal cytology findings are rare in patients with radiologically resectable adenocarcinoma of the pancreas. When found, positive peritoneal washings are an indicator of advanced disease characterized by unresectability, early metastasis, and short survival. Computed tomographic-guided FNA does not appear to increase the risk for positive peritoneal washings and represents a valid approach to the pretreatment diagnosis of patients with suspected pancreatic malignancy.

Adenocarcinoma↗
PubMed1994

Site-directed mutagenesis of adeno-associated virus type 2 structural protein initiation codons: effects on regulation of synthesis and biological activity.

It has been shown that two of the three adeno-associated virus type 2 capsid proteins, B and C, are synthesized from a single spliced transcript. Protein C arises from an AUG codon at nucleotide 2810, whereas protein B is initiated by a unique eucaryotic initiation codon (ACG) that lies 65 triplets upstream from the C origin. The third capsid component, protein A, is synthesized from a second spliced transcript which uses an alternative 3' acceptor site. In this study we used oligonucleotide-directed mutagenesis to confirm the positions of the B initiation codon and the 3' acceptor sites for the alternatively spliced B/C and A protein messages. We also located definitively the protein A initiation codon, an AUG triplet mapping to nucleotide 2203. Mutagenesis of the B initiator permitted a direct test of the effect of increased B initiator strength on the translational efficiencies of the B and C proteins. It was found that conversion of the relatively inefficient protein B initiator (ACG) to an AUG enhanced the level of B synthesis while abolishing the synthesis of C from its downstream AUG initiator. Protein C synthesis thus depends on the strength of the B initiator, i.e., the relatively higher levels of C (approximately 20-fold greater than B) must result from frequent readthrough of the weak B initiator. Finally, we examined the abilities of mutants deficient in the synthesis of A, B, or C to produce infectious virions. We found that at least two of the structural proteins, B and C, are required for the production of infectious virions and that sequestration of single-stranded adeno-associated virus genomes from the pool of replicating DNA molecules does not occur in the absence of either of these proteins.

Blotting, Northern↗
PubMed1992

Arterial norepinephrine changes in patients with septic shock.

Arterial, mixed venous (pulmonary arterial), and peripheral venous norepinephrine and epinephrine levels; hemodynamics; and blood lactate levels were measured in 28 patients with septic shock (16 men and 12 women). During hospital follow-up, 18 patients (64%) died of circulatory failure. There were no significant differences in hemodynamic parameters or initial blood lactate levels between survivors and nonsurvivors. Initial arterial, mixed venous, and peripheral venous norepinephrine levels were elevated above normal in both survivors and nonsurvivors. However, norepinephrine levels at all three sampling sites were significantly higher in nonsurvivors than in survivors. Arterial or mixed venous norepinephrine level was better than peripheral venous norepinephrine level in distinguishing survivors from nonsurvivors. In contrast, the differences in plasma epinephrine levels between survivors and nonsurvivors became significantly different only after 48 hr of follow-up. During 60 degrees head-up tilt, the increase in plasma norepinephrine level was significantly higher in survivors compared to non-survivors, suggesting a differential response in the sympathetic nervous system in the two groups of patients. These data suggest that measurement of arterial or mixed venous plasma norepinephrine levels may be a useful guide for assessing the clinical course of patients in septic shock. Moreover, the differences in the sympathetic nervous system response to a 60 degree tilt may predict a poor outcome in these patients.

Acidosis, Lactic↗
PubMed1990

Adeno-associated virus DNA replication is induced by genes that are essential for HSV-1 DNA synthesis.

Adeno-associated virus (AAV) DNA replication is not detectable unless cells are coinfected with a helper adenovirus (Ad) or herpesvirus or unless AAV infection is carried out in certain established cell lines that have been treated with various metabolic inhibitors or uv irradiation. In helper-dependent infections, it has been shown that AAV DNA synthesis depends on one or more early Ad genes, whereas little is known concerning any herpesvirus gene that promotes AAV DNA synthesis. In this study we tested the ability of four cloned Xbal fragments of herpes simplex virus type 1 (HSV-1) DNA to induce AAV DNA synthesis in Vero cells. Cotransfections, which were carried out with pAV1 (an infectious AAV2 plasmid), revealed that AAV DNA synthesis could be optimally induced by three of these clones (C,D, and F) plus a clone of the HSV-1 ICP4 (IE 175) gene. ICP4, an immediate early gene, was presumably required to activate expression of other HSV genes. To help identify the additionally needed HSV genes, we tested Xbal C,D, and F subclones that contain genes previously found necessary for origin-dependent HSV DNA synthesis and found that at least five of these genes (UL 5, 8, 9, 29, and 30) contributed to the induction of AAV DNA synthesis. In contrast to their absolute requirement for HSV DNA synthesis, none of these genes were strictly necessary for AAV DNA replication. Because they are all known to specify proteins that are directly involved in HSV DNA synthesis, our results suggest that some or all of their products also may directly participate in the replication of AAV DNA.

Animals↗
PubMed1989

Efficient synthesis of adeno-associated virus structural proteins requires both adenovirus DNA binding protein and VA I RNA.

We have shown previously that replication of defective parvoviruses [adeno-associated viruses (AAV)] requires several early adenovirus (Ad) gene products [J. E. Janik, M. M. Huston, and J. A. Rose (1981) Proc. Natl. Acad. Sci. USA 78, 1925-1929]. To examine their possible roles in the transcription and translation of AAV mRNA, 293-31 cells, a human embryonic kidney cell line that constitutively expresses the Ad early region IA and IB gene products, were transfected with a pBR325 plasmid (pLH1) that contains a duplex AAV2 DNA segment (0.03-0.97 map units) which encompasses the promoters and coding sequences necessary for expression of all AAV polypeptides. When cells were transfected with pLH1 alone, both spliced and unspliced AAV-specific cytoplasmic RNAs accumulated. These transcripts were capable of directing synthesis of the three AAV capsid polypeptides in vitro, whereas in vivo synthesis of AAV protein was not detected by immunofluorescence or immunoprecipitation. When cells were cotransfected with pLH1 and intact Ad DNA, the level of cytoplasmic AAV RNA was enhanced and AAV protein was synthesized in vivo. Additional experiments demonstrated that in vivo AAV protein synthesis also could be induced when pLH1 was cotransfected with plasmids that contain the Ad DNA-binding protein (pDBP) and VA I RNA (p2BalM) genes; however, a low level of in vivo AAV capsid protein was occasionally detected in cotransfections with pLH1 and a plasmid that contains both VA I and VA II RNA coding sequences (p2SalC). Cotransfection of pLH1 and pDBP or pLH1 and p2SalC showed complex alterations in the steady-state patterns of AAV cytoplasmic transcripts. In both cases, increased levels of transcripts, particularly the 2.3-kb spliced species, were detected in comparison to levels seen in cells transfected with pLH1 alone. Despite these increases, however, there was little, if any, induction of AAV protein synthesis unless both the DNA-binding protein (DBP) and VA I RNA coding sequences were present in cotransfection with pLH1. We conclude that, in 293-31 cells, the Ad VA I RNA and DBP gene products regulate AAV capsid protein synthesis at least at two levels: (i) by increasing the steady-state levels of structural protein transcripts in the cytoplasm, especially the spliced species, and (ii) by enhancing the translation of these messages.

Capsid↗
PubMed1988

Synthesis of adeno-associated virus structural proteins requires both alternative mRNA splicing and alternative initiations from a single transcript.

The three adeno-associated virus type 2 (AAV2) structural proteins (A, B, and C) are specified by transcripts generated from the most-rightward promoter (p40). Protein C (60 kilodaltons [kDa]), the most abundantly produced, is entirely contained within B (72 kDa) which, in turn, is contained within A (90 kDa). Although neither of the known structures of p40 transcripts, an unspliced 2.6-kilobase (kb) RNA and a spliced 2.3-kb RNA, possesses an AUG-initiated open reading frame that accounts for the synthesis of proteins A and B, recent evidence indicates that B is initiated by a unique eucaryotic initiation codon (ACG) (S.P. Becerra, J.A. Rose, M. Hardy, B. Baroudy, and C.W. Anderson, Proc. Natl. Acad. Sci. USA 82:7919-7923, 1985). In the present study, we analyzed the in vitro translation of AAV capsid proteins from synthetic transcripts and the in vivo expression of AAV mRNA and capsid proteins in 293 cells transfected with AAV DNA constructs. The results demonstrated that AAV transcripts contain only one functional 5' splice donor site, that synthesis of capsid proteins from the unspliced 2.6-kb transcript is very inefficient, that transcripts without the intervening sequence (IVS) (i.e., the 2.3-kb RNA) do not produce protein A but effectively synthesize proteins B and C, and that protein A is actively synthesized from transcripts which contain the last 34 bases of the IVS. Protein A initiates within this 34-base segment in reading frame 1, apparently with the AUG codon at nucleotide 2203, and then elongates into the B and C open reading frame. Because A is inefficiently synthesized from the 2.6-kb transcript, we conclude that an effective A transcript is generated by alternative splicing and that the alternative 3' acceptor site may lie at nucleotide 2200 within a context of...CAG]GTA. The levels of B and C produced by a synthetic transcript devoid of the IVS suggest that the known 2.3-kb RNA is the main source of these proteins and indicate that this single RNA species expresses both proteins by alternative use of their respective initiation codons.

Capsid↗
PubMed1985

Human cytomegalovirus completely helps adeno-associated virus replication.

Coinfection of adeno-associated virus (AAV) with human cytomegalovirus (HCMV) strain Towne in human embryonic fibroblasts resulted in accumulation of AAV capsid antigen and production of infectious AAV with a lag of 24 hr compared to AAV replication in AAV-adenovirus coinfections. In contrast to previous observation, these findings demonstrated that HCMV is a competent helper virus for the complete replication of AAV. In addition, HCMV and AAV were synergistic in their cytopathic effects on cells, suggesting the possibility that AAV may play a role in the pathogenicity of HCMV infections.

Antigens, Viral↗
PubMed1985

Direct mapping of adeno-associated virus capsid proteins B and C: a possible ACG initiation codon.

The three major capsid proteins of adeno-associated virus type 2 (AAV2) virions are designated A, B, and C and have molecular sizes of 90, 72, and 60 kDa, respectively. These proteins are related, and genetic studies have shown they are encoded by a long open reading frame located in the right half of the genome. The coding capacity distal to the first ATG in this reading frame is only 503 amino acids (i.e., a protein about the size of protein C), but an open frame sequence devoid of ATG codons extends upstream for an additional 184 codons. Although the amino terminus of the C capsid protein is blocked, partial amino acid sequence analyses of peptides from C have confirmed that it is encoded within the portion of the reading frame distal to the first ATG at nucleotide (nt) location 2810. The amino terminus of the B capsid protein is not blocked, and its sequence begins with alanine. The triplet encoding this alanine lies 64 codons upstream from the initiation site for protein C and is immediately preceded by the threonine codon, ACG, at nt 2615. This ACG codon lies in the most favorable sequence context for protein synthesis initiation. All three AAV2 capsid proteins are labeled in vitro with formyl[35S]methionyl-tRNAf, indicating that synthesis of each protein is initiated independently. Our data suggest that the nt 2615 ACG codon directs the methionyl-tRNA-dependent initiation of the AAV2 B capsid protein. Proteins B and C may be synthesized from the same mRNA species and their relative abundance could be determined by the efficiencies of their respective initiation codons.

Amino Acid Sequence↗
PubMed1984

Adeno-associated virus proteins: origin of the capsid components.

The three primary capsid proteins (A, B, and C) of adeno-associated viruses have been shown previously to contain overlapping amino acid sequences (R. McPherson and J. Rose, J. Virol. 46:523-529, 1983). In the present study we demonstrate definitively that these proteins are encoded in the right half of the adeno-associated virus 2 genome, and one or both of the smallest adeno-associated RNA species (2.3- or 2.6-kilobase RNA) account for their synthesis. Protein A (90 kilodaltons) apparently initiates from a site within the intervening sequence, which is intact in the larger (unspliced) 2.6-kilobase mRNA, and may read through one or more termination codons, including a strong stop signal (UAA) that lies 31 bases downstream from the end of the intervening sequence. Proteins B (72 kilodaltons) and C (60 kilodaltons) are not derived from protein A but apparently originate from independent, in-frame initiations that lie downstream from the splice junction. It thus seems likely that production of the three adeno-associated virus capsid proteins involves at least two mRNA species. The B and C proteins presumably arise from the spliced 2.3-kilobase RNA, whereas protein A should be generated by the 2.6-kilobase RNA or a hitherto unidentified spliced RNA species.

Base Sequence↗
PubMed1983

Structural proteins of adenovirus-associated virus: subspecies and their relatedness.

Capsids of adenovirus-associated virus (AAV) are known to contain three major structural proteins (A, B, and C). We have further resolved distinct subspecies of two of the major AAV proteins (two forms of protein A and four forms of protein C) which were found in both AAV1 and AAV2 serotypes. All subspecies were accurately synthesized in a cell-free translation system programmed with RNA isolated from infected cells. Analysis of virion proteins from the autonomous parvovirus H1 did not reveal a comparable array of subspecies of its major components. Staphylococcal V8 protease digestion of C proteins from AAV1 and AAV2 yielded very different electrophoretic patterns, indicating a considerable difference between the C proteins of these two serotypes, despite a high degree of genomic homology and an overall similarity in the number and relative proportions of analogous capsid proteins. On the other hand, staphylococcal V8 protease digestion of isolated proteins A, B, and C of AAV2 showed an extensive overlap among these proteins, possibly equivalent to all of protein C. In conjunction with other data, these findings suggest that proteins A, B, and C arise from different in-frame initiation sites contained in mRNA sequences that are transcribed from the right half of the AAV genome. The heterogeneity of subspecies may be explained by a partial read through of several tandem termination codons near the 3' end of AAV mRNA.

Dependovirus↗
PubMed1982

Adeno-associated virus helper activity of adenovirus DNA binding protein.

The requirement for the adenovirus (Ad) single-stranded DNA binding protein (DBP) in the expression of adeno-associated virus (AAV) proteins was studied by specific immunofluorescent staining of infected cells and in vitro translation of RNA from infected cells. The Ad5 mutant ts125, which carries a mutation in the DBP gene, helped AAV as efficiently as the Ad5 wild type (WT) did at both the permissive (32 degrees C) and nonpermissive (40.5 degrees C) temperatures in HeLa and KB cells. Furthermore, at 40.5 degrees C ts125 was as efficient as Ad5WT was in inducing the expression of AAV proteins in a line of Detroit 6 cells which is latently infected with AAV. However, little if any AAV protein was synthesized when coinfections were carried out with Ad5WT in CV-C cells, a monkey cell line that is highly restrictive for human Ad replication unless the cells are also infected with simian virus 40. On the other hand, AAV protein was efficiently produced in CV-C cells in coinfections with the Ad5 mutant hr404, whose growth is unrestricted in CV-C cells and whose mutation also maps in the DBP gene. Finally, preparations of cytoplasmic RNA extracted from CV-C cells infected with AAV and Ad5WT or from CV-C cells infected with AAV, Ad5WT, and simian virus 40 were each capable of directing the in vitro synthesis of abundant amounts of AAV proteins in a rabbit reticulocyte lysate system. These results indicate that the abnormal DBP of ts125 still retains its helper function for AAV replication, but that the molecular feature of the DBP which relates to the monkey cell host range restriction of Ad's may also account for the observed block to AAV protein translation in CV-C cells.

Adenoviruses, Human↗
PubMed1982

Comparison of twice-daily ranitidine and placebo in the treatment of duodenal ulcer--a multicentre study in the United Kingdom.

Treatment of duodenal ulcer with the H2-receptor antagonist ranitidine, 150 mg twice daily has been assessed in a double-blind, placebo controlled study at seven centres in the United Kingdom. One hundred-and-twenty-nine patients entered the trial with endoscopically confirmed duodenal ulcer. Five patients did not comply with the protocol and were excluded from the analysis. Nine patients (1 ranitidine, 8 placebo) did not complete the initial 4 weeks' treatment due to poor symptomatic response; one hundred-and-fifteen (58 ranitidine, 57 placebo) were endoscopically assessed after 4 weeks. The average 4-week healing rate among patients on ranitidine (83%) was significantly greater than that for the placebo group (32%, p less than 0.01). Forty-four patients whose ulcers had not healed received further treatment with ranitidine 150 mg b.d. on an open basis. After a total of up to 8 weeks' active treatment only three patients had not healed. Ulcer symptoms resolved or improved in a greater proportion of patients on ranitidine, and this was associated with a significantly lower antacid consumption. There was no serious unwanted effect associated with ranitidine treatment, and the twice daily dose of 150 mg is apparently a safe and effective short-term treatment for duodenal ulceration.

Adult↗
PubMed1981

Deoxyribonucleic acid single-strand-specific endonucleases in human cells: partial purification of a salt-resistant endonuclease with an acidic isoelectric point.

A second endonuclease with DNA single-strand specificity has been purified from KB cells, a continuous line of hunan epithelial cells. In contrast to other mammalian enzymes that cleave single-stranded DNA, this enzyme has an acidic isoelectric point (6.5 +/- 0.2). Its pH optimum is 9.5, it requires Mg2+ of Mn2+ for activity, and it has a sedimentation coefficient of 3.2 S, based on sucrose gradient centrifugation. The enzyme specifically catalyzes the endonucleolytic cleavage of synthetic DNA homopolymers and denatured viral DNA but does not attack linear duplex viral DNA. The rate of hydrolysis of poly(dT) is approximately 8-fold greater than that observed with denatured DNA. The relative rates of hydrolysis of homopolymers by the endonuclease are poly(dA) greater than poly(dT) greater than poly(dC) greater than poly(dG). Unlike other DNA single-strand-specific endonucleases isolated from human cells, this endonuclease is relatively insensitive to inhibition by KCl.

Cell Line↗
PubMed1981

Locations of adenovirus genes required for the replication of adenovirus-associated virus.

We have used DNA transfection to identify several regions of the adenovirus genome needed to induce replication of the defective parvovirus, adenovirus-associated virus (AAV). Previous studies have indicated that only early adenovirus functions are needed to aid the replication of AAV. In this report, we demonstrate that three restriction endonuclease fragments of adenovirus DNA are necessary for production of infectious AAV in 293-31 cells (an adenovirus type 5-transformed human embryonic kidney cell line). These fragments map from 28.5 to 29.4, 59.5 to 75.9, and 89.7 to 100 map units on the adenovirus type 2 genome and correspond to the locations of the VAI RNA gene, early region 2, and early region 4, respectively. The 293-31 cell line, which has been found to express early region 1A and 1B proteins, alone is incapable of supporting AAV replication or even AAV DNA synthesis. Additional experiments with adenovirus type 5 host range mutants (group I, hr1 and group II, hr7) indicate, however, that early region 1A provides an essential function(s) for AAV replication, whereas early region 1B probably does not.

Adenoviruses, Human↗
PubMed1981

Herpes simplex virus types 1 and 2 completely help adenovirus-associated virus replication.

In addition to adenoviruses, which are capable of completely helping adenovirus-associated virus (AAV) multiplication, only herpesviruses are known to provide any AAV helper activity, but this activity has been thought to be partial (i.e., AAV DNA, RNA, and protein syntheses are induced, but infectious particles are not assembled). In this study, however, we show that herpes simplex virus type 1 (HSV-1) and type 2 (HSV-2) are in fact complete AAV helpers and that AAV type 2 (AAV2) infectivity yields can approach those obtained when coinfections are carried out with a helper adenovirus. AAV helper activity was demonstrated in KB cells with two HSV-1 strains (11124 and 17MP) and an HSV-2 strain (HG52). Each herpesvirus supported AAV2 multiplication with comparable efficiency. AAV2 multiplication was similarly efficient in HSV-1 coinfections of HeLa cells, whereas lower yields were obtained in HEp-2 and primary human embryonic kidney cells. HSV-1 also supported AAV1 multiplication in HeLa cells but, at corresponding multiplicities of infection, AAV1 grew less efficiently than AAV2. Comparisons of the time courses of AAV2 DNA, RNA, and protein syntheses after coinfection with either adenovirus type 5 or HSV-1 revealed that, in each case, the onset of synthesis and attainment of maximal synthesis rate occurred earlier in coinfections with HSV-1. These findings demonstrate the linkage of AAV macromolecular synthesis to an event(s) in the helper virus cycle. Aside from this temporal association, helper-related differences in AAV macromolecular synthesis were not apparent.

DNA, Viral↗
PubMed1979

Mechanism of host restriction of adenovirus-associated virus replication in African green monkey kidney cells.

Human adenovirus (Ad) serotypes provide an early factor(s) that is necessary for adenovirus-associated virus (AAV) multiplication in human cell lines. However, little, if any, AAV production occurs in primary African green monkey kidney (AGMK) cells co-infected with AAV and a helper human Ad (non-permissive infection), unless cells are additionally infected with SV40 (permissive infection). To determine the basis of the host restriction of AAV replication in AGMK cells, AAV DNA, RNA and protein synthesis were analyzed under various conditions of infection. Hybridization reactions revealed no detectable AAV-specific DNA or RNA in infections with AAV alone or in combination with SV40. In co-infections with AAV and Ad5 or Ad7, the synthesis of both AAV- and Ad-specific DNA and RNA occurred without a significant rise in titre of either virus. During non-permissive infection, however, AAV DNA synthesis was abnormal in that an expected accumulation of single-stranded progeny molecules was not observed. Finally, although intact 20S AAV transcripts were present in the cytoplasm of AGMK cells during non-permissive infection (in amounts ranging from 50 to 80% of that found during permissive infection), AAV-specific polypeptides were not demonstrable by polyacrylamide gel electrophoresis. Taken together, these experiments indicate that the host restriction of AAV replication in AGMK cells is exerted at the level of translation of the single AAV messenger RNA. In addition, it appears that one or more of the AAV polypeptides specified by this message is required for the production of single-stranded AAV progeny DNA.

Adenoviruses, Human↗