Search PubMed⌕ Search

Biomedical subjects

L K Miller

Publications and source records attributed to L K Miller.

At least 91 records · Page 5Linked to original sources
PubMed1993

Multinodular goiter of the thyroid mimicking malignancy: diagnostic pitfalls in fine-needle aspiration biopsy.

A case of multinodular goiter with multicentric papillary hyperplasia is presented with cytologic features suggestive of thyroid papillary carcinoma. While the cytologic criteria for papillary thyroid carcinoma is well defined, occasional diagnostic difficulties can arise. Cytologically, the presence of focal papillary aggregates, the presence of a psammoma body within a background of copious colloid and scattered follicular cells led to diagnostic confusion. Histologically, the presence of secondary follicles in the papillae and protrusion of the papillary structures into colloid lakes supported the diagnosis of goiter. It is our opinion that most, if not all, of these difficulties can be circumvented by the incorporation of all major and some minor cytologic criteria for papillary carcinoma in the analysis of such lesions. Single criteria for papillary carcinoma, either major or minor, is not sufficient.

Aged↗
PubMed1993

Characterization of productive and non-productive AcMNPV infection in selected insect cell lines.

We have investigated the process of productive and non-productive AcMNPV infection in a variety of cultured insect cells including lines derived from Spodoptera frugiperda (IPLB-SF21), Mamestra brassicae (SES-MaBr-3), Choristoneura fumiferana (IPRL-CF-1), Bombyx mori (BmN-4), Lymantria dispar (IPLB-Ld652Y), Helicoverpa zea (Hz1b3), and Drosophila melanogaster (Dm). In each cell line, we have examined viral utilization of an early, a late, and a very late promoter, the replication of viral DNA and the production of budded virus (BV) and polyhedral inclusion bodies (PIBs). Analysis of promoter use on the single cell level was performed using fluorescence-activated cell sorting (FACS) technology. Cell cultures were infected with recombinant viruses containing CAT reporter genes under the transcriptional control of viral early, late or very late promoters. Cells were immunostained to detect the CAT gene product and the relative CAT content of infected cells on a per cell basis was determined by FACS analysis. Productive infection of IPLB-SF21 cells involved essentially 100% of the cells while analysis of less productive lines (IPRL-CF-1 and SES-MaBr-3) suggests that the virus initiates and completes the replication process only in a subpopulation of cells. Analysis of non-productive infections (in BmN-4, IPLB-Ld652Y, Hz1b3, Drosophila Schneider) revealed different patterns of viral DNA replication and promoter use in each cell line suggesting a variety of obstacles to productive infection.

Animals↗
PubMed1993

Identification of genes encoding late expression factors located between 56.0 and 65.4 map units of the Autographa californica nuclear polyhedrosis virus genome.

Using a previously developed method that allows the identification of Autographa californica nuclear polyhedrosis virus (AcMNPV) genes which stimulate transient expression from a late and a very late viral promoter [Passarelli and Miller, J. Virol., 67, 2149-2158 (1993)], we have identified three genes between 56.0 and 65.4 map units of the AcMNPV genome involved in expression from a late and a very late promoter but not from an early viral promoter. One gene, p143, was previously shown to be essential for viral DNA replication and shares sequence motifs with DNA helicases [Lu and Carstens, Virology, 181, 336-347 (1991)]. The second gene, previously sequenced and originally referred to as open reading frame 6 (herein renamed late expression factor-5 [lef-5]), was located just downstream of the 6.9 kilodalton core protein gene, p6.9. The third gene, late expression factor-4 (lef-4), was defined and sequenced. The lef-4 gene was located immediately upstream of, and in opposite orientation to, the major capsid protein gene, vp39. The position and direction of lef-4 appeared to be conserved in the Orgyia pseudotsugata and Lymantria dispar nuclear polyhedrosis viruses. The gene product of lef-4, LEF-4, is predicted to be an acidic polypeptide (pl 4.91) of 464 amino acids in length with a molecular mass of 53,913 daltons.

Amino Acid Sequence↗
PubMed1993

Identification and characterization of tox21A: a mite cDNA encoding a paralytic neurotoxin related to TxP-I.

A cDNA library established from polyadenylated RNA isolated from the predatory mite Pyemotes tritici was screened for cDNAs homologous to tox34, a cDNA encoding an insect-selective paralytic neurotoxin known as TxP-I. Most of the cDNA inserts of homologous clones were shorter or of equal length to tox34 but a few were longer. Further investigation into the nature of these longer clones led to the identification, sequencing and expression of a distinct cDNA referred to as tox21A. This cDNA is predicted to encode a polypeptide which shares approximately 83% amino acid identity with TxP-I. Larvae infected with a recombinant baculovirus expressing tox21A are paralyzed during infection in a manner similar to larvae infected with tox34-expressing recombinants. The tox21A cDNA may represent a duplicated and diverged copy of the TxP-I gene.

Amino Acid Sequence↗
PubMed1993

Baculoviruses: high-level expression in insect cells.

Baculoviruses continue to serve as workhorse vectors for the high-level expression of eukaryotic genes in insect cells; however, numerous researchers are also finding novel uses for these vectors by taking advantage of the unique nature of the viruses and their host cells. Concurrently, the technology involved in constructing and utilizing these vectors is being improved so that the time and effort required to construct expression vectors are reduced.

Animals↗
PubMed1993

Three baculovirus genes involved in late and very late gene expression: ie-1, ie-n, and lef-2.

We developed a method to identify baculovirus genes required for late and very late gene expression that is based on subtraction of clones from an Autographa californica nuclear polyhedrosis virus genomic library which is able to trans activate promoters of reporter plasmids in transient expression assays. Using this assay, we found that three genes located between 83.7 and 7.5 map units of the Autographa californica nuclear polyhedrosis virus genome were involved in expression from the late capsid protein (vp39) and very late polyhedrin (polh) gene promoters. Two of these genes, ie-1 and ie-n, trans regulate early genes in transient expression assays. Although ie-1 was necessary and sufficient for expression from the early promoter in our assay, it was necessary but not sufficient for expression from the vp39 and polh promoters. The presence of ie-n increased expression from the early, late, and very late classes of promoters tested. However, a third gene identified in this region was specifically required for expression from the vp39 and polh promoters. This gene, a previously sequenced 630-nucleotide open reading frame, was renamed lef-2 for late expression factor 2. We also found that other genes in the region between 83.7 and 7.5 map units were not required for expression from the promoters used in this assay, although we did not eliminate the possibility that they subtly modify expression. These genes include pe-38 and me53, early genes with zinc finger-like motifs, and the upstream exon of ie-0, which specifies an alternate form of IE-1.

Animals↗
PubMed1993

An apoptosis-inhibiting baculovirus gene with a zinc finger-like motif.

Spodoptera frugiperda SF-21 cells infected with Autographa californica nuclear polyhedrosis virus mutants which lack a functional p35 gene undergo apoptosis, a type of programmed cell death. To identify p35-homologous genes in other baculoviruses, A. californica nuclear polyhedrosis virus DNA containing a deletion in p35 was cotransfected into SF-21 cells along with genomic DNAs from other baculoviruses. One of the viral DNAs which were able to rescue wild-type infection was from Cydia pomonella granulosis virus (CpGV). The CpGV gene responsible for the effect was mapped to a 1.6-kb SalI-SstI subclone of the SalI B fragment of CpGV. The sequence of the SalI-SstI subclone revealed an open reading frame capable of encoding a polypeptide of 31 kDa which was sufficient to rescue wild-type infection; this gene was thus called iap (inhibitor of apoptosis). The predicted sequence of the IAP polypeptide exhibited no significant homology to P35 but contained a zinc finger-like motif which is also found in other genes with the potential to regulate apoptosis, including several mammalian proto-oncogenes and two insect genes involved in embryonic development. In the context of the viral genome, both iap and p35 were able to block apoptosis induced by actinomycin D, indicating that these genes act by blocking cellular apoptosis rather than by preventing viral stimulation of apoptosis. Several independent recombinant viruses derived from cotransfections with either the entire CpGV genome or the 1.6-kb subclone were characterized.

Amino Acid Sequence↗
PubMed1993

Identification and characterization of lef-1, a baculovirus gene involved in late and very late gene expression.

An Autographa californica nuclear polyhedrosis virus (AcMNPV) gene required in transient expression assays for late and very late viral gene expression was identified, sequenced, and transcriptionally mapped. This gene, designated late expression factor 1 (lef-1), was located between 7.4 and 8.7 map units of the AcMNPV physical map. It was identified by cotransfecting Spodoptera frugiperda cultured cells with a collection of overlapping cloned DNA fragments covering the entire AcMNPV genome and a reporter gene controlled by an early, late, or very late AcMNPV promoter. Omission of the DNA fragment containing lef-1 curtailed most late and very late gene expression but not early gene expression. lef-1 was found to be an early gene transcribed as a 1.8-kb RNA in the presence of the protein synthesis inhibitor cycloheximide. The C terminus of the predicted polypeptide product, LEF-1, contained a sequence motif characteristic of nucleoside triphosphate-binding sites.

Amino Acid Sequence↗
PubMed1993

Apoptosis reduces both the in vitro replication and the in vivo infectivity of a baculovirus.

Apoptotic programmed cell death occurs when the insect cell line SF-21, derived from Spodoptera frugiperda, is infected with mutants of the baculovirus Autographa californica nuclear polyhedrosis virus (AcMNPV) which lack a functional p35 gene. However, infection of the Trichoplusia ni TN-368 cell line with p35 mutants does not result in apoptosis (R. Clem, M. Fechheimer, and L. Miller, Science 254:1388-1390, 1991). We have examined the effect of apoptosis on AcMNPV infections in cell lines and larvae of these two insect species. Production of viral progeny was significantly lower in SF-21 cells infected with p35 mutants than in cells infected with wild-type (wt) or revertant viruses. Viral gene expression was abnormal in SF-21 cells infected with p35 mutants; there was a delay in the transcription and translation of early and late viral genes, a lack of expression of very late genes, and a total cessation of protein synthesis late in the apoptotic process. In vivo analysis revealed that the dose of budded virus required for 50% lethality in S. frugiperda larvae was approximately 1,000-fold higher for p35 mutants than for wt or revertant viruses. In contrast, the replication and infectivity of p35 mutant viruses was equivalent to that of wt AcMNPV during infection of both TN-368 cells and T. ni larvae. Thus, the data indicate that a host apoptotic response provides protection against viral infection at the organismal level and that the p35 gene constitutes a host range determinant for AcMNPV infection.

Animals↗
PubMed1993

Identification, sequence, and transcriptional mapping of lef-3, a baculovirus gene involved in late and very late gene expression.

A trans-acting gene required for late viral gene expression in transient expression assays was identified in the genome of Autographa californica nuclear polyhedrosis virus. A genomic library of A. californica nuclear polyhedrosis virus DNA lacking a clone spanning the region from 43 to 48 map units (mu) was unable to activate gene expression from a reporter plasmid when the reporter gene was driven by a baculovirus late or very late promoter in transient expression assays. The genomic region responsible for activating reporter gene expression was further mapped to 43.4 to 45.2 mu of the viral genome. The nucleotide sequence of this region was determined and shown to contain several small open reading frames (ORFs) and one major ORF, named lef-3, for late expression factor 3. The lef-3 ORF was predicted to encode a polypeptide of 385 amino acids and with a molecular mass of 44,529 daltons. No homolog to the lef-3 ORF was found in existing data bases. Further analysis showed that only the lef-3 ORF in the region from 43.4 to 45.2 mu was necessary for late gene activation in the assays used. The temporal regulation of lef-3 transcription was studied by Northern (RNA) blot hybridization; lef-3 was found to be an early gene that was transcribed primarily as a 2.0-kb mRNA. Primer extension analysis and S1 nuclease protection assays revealed that lef-3 transcription initiated about 280 bp upstream of the first ATG codon and terminated near a polyadenylation signal, 130 bp downstream of the last codon of the lef-3 ORF.

Amino Acid Sequence↗
PubMed1993

Efficacy and maintenance of an education program for a consumer cooperative.

We examined the effects of contingency management on participation in and maintenance of an education program by new members of a student housing cooperative. With credit and fine contingencies in place, the percentage of participants completing study guides was five times higher than without the contingencies. Members continued to implement the program for 9 years without researcher involvement.

Adolescent↗
PubMed1992

HIV-associated oral lesions; immunologic, virologic and salivary parameters.

There are numerous reports of oral lesions in HIV-infected individuals. However, few correlate the oral lesions with laboratory parameters. This study examined oral candidiasis and hairy leukoplakia, the two most common HIV-associated oral lesions, in relation to T-cell counts, p24 core antigen levels and salivary flow rates. Oral mucosal examinations, immunologic and virologic studies and stimulated whole and parotid saliva flow rates were conducted on 135 (HIV+ = 102, HIV- = 33) homosexual or bisexual men. Results indicate that, among HIV-infected subjects, the odds of having oral candidiasis is 6 times (95% CI = 0.6-56.6) greater for subjects with T4 counts between 200-399 per mm3, and 23 times (95% CI = 2.8-193.0) greater for subjects with T4 counts less than 200/mm3 compared to subjects with T4 counts of 400/mm3 or greater. Subjects had an equal likelihood of having hairy leukoplakia at different levels of immunocompetence. The prevalence of oral candidiasis and hairy leukoplakia was higher among subjects with infectious virus in their serum, but was only statistically significant for hairy leukoplakia (p = 0.01).

Adult↗
PubMed1992

Insecticidal properties of genetically engineered baculoviruses expressing an insect juvenile hormone esterase gene.

Exploring the possibility of enhancing the properties of baculoviruses as biological control agents of insect pests, we tested the effect of expressing an insect gene (jhe) encoding juvenile hormone esterase. Juvenile hormone esterase inactivates juvenile hormone, which regulates the outcome of an insect molt. A cDNA encoding the juvenile hormone esterase of Heliothis virescens was inserted into the genome of Autographa californica nuclear polyhedrosis virus such that the gene was expressed under the control of a strong, modified viral promoter. This virus, however, naturally encodes an ecdysteroid UDP-glucosyltransferase which inactivates ecdysone, the hormone which initiates molting. Since ecdysteroid UDP-glucosyltransferase could mask the effects of jhe expression by blocking molting entirely, jhe-expressing viruses in which the ecdysteroid UDP-glucosyltransferase gene was deleted or disrupted were constructed. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of proteins from infected cells revealed several intracellular proteins and two major secreted proteins which reacted with antibodies to authentic juvenile hormone esterase. Western blot analysis coupled with tunicamycin treatment indicated that differential glycosylation was responsible for the multiple products. Hemolymph of recombinant virus-infected fourth-instar Trichoplusia ni larvae contained levels of juvenile hormone esterase activity 40-fold higher than maximal levels found in uninfected larvae. However, little or no difference in developmental characteristics, weight gain, or time of mortality was observed between insects infected with the jhe-expressing viruses and control viruses.

Animals↗
PubMed1992

Characterization of a baculovirus gene encoding a small conotoxinlike polypeptide.

We identified a gene of Autographa californica nuclear polyhedrosis virus (AcMNPV) that encodes a small cysteine-rich polypeptide which has size and sequence similarity to omega-conotoxins, a class of calcium ion (Ca2+) channel inhibitors, found in the venom of cone snails. Transcriptional analysis indicated that the 159-bp open reading frame, which we named ctl, and a downstream 984-bp open reading frame are transcribed as a single 1.3-kb bicistronic late RNA. The mature ctl gene product was identified as a small secreted protein by high-pressure liquid chromatography fractionation of extracellular fluid. Viruses with a site-specific deletion in ctl appeared normal with regard to the kinetics and virulence of infection, both in vitro and in vivo. Although we studied the behavior of wild-type and mutant virus-infected insects in some detail, a biological role for ctl in AcMNPV infection remains to be established.

Amino Acid Sequence↗
PubMed1992

Promoter influence on baculovirus-mediated gene expression in permissive and nonpermissive insect cell lines.

The activities of viral and insect promoters were examined in a range of insect cell lines permissive and nonpermissive for the replication of the baculovirus Autographa californica nuclear polyhedrosis virus. Recombinant baculoviruses were constructed to place the bacterial chloramphenicol acetyltransferase gene under the control of promoters strongly active in the early, late, or very late stages of virus replication. In fully permissive cells, expression from a very late promoter was 2- to 3-fold higher than expression from a late promoter and 10- to 20-fold higher than expression from an early promoter or from a virus-borne insect promoter. In cell lines that do not support the efficient production of viral progeny, late-promoter-driven expression was similar to or surpassed very late promoter-driven expression. In nonpermissive insect cell lines, expression driven by an insect promoter derived from Drosophila melanogaster was higher than expression from the three viral promoters and was especially high in the Drosophila cell line tested. Surprisingly, late-promoter-driven expression, which is dependent on DNA replication, was higher than early-promoter-driven expression in three of four nonpermissive lines. In contrast, very late promoter-driven expression was quite limited in nonpermissive cell lines. The results indicate that the promoter used to drive foreign-gene expression strongly influences the range of insect cells which can efficiently support the production of the foreign protein during infection with recombinant baculoviruses.

Animals↗
PubMed1992

Recovery of infectious HIV-1 from whole saliva.

Whole saliva and serum samples were collected from 75 HIV-infected homosexual or bisexual men. Thirty-eight percent of cultured sera were positive for cell-free, infectious virus while only 1 percent of the 218 cultured whole salivas contained cell-free, infectious virus. These data support previous studies suggesting unlikely potential transmissibility of HIV infection by saliva.

Adult↗