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L K Miller

Publications and source records attributed to L K Miller.

At least 73 records · Page 4Linked to original sources
PubMed1995

Properties of a baculovirus mutant defective in the protein phosphatase gene.

Autographa california nuclear polyhedrosis virus (AcMNPV) contains a gene, ptp, encoding a protein tyrosine/serine phosphatase, BV-PTP. To investigate the biological function of ptp in the baculoviral replication cycle, we constructed a recombinant baculovirus, vPTPdel, in which the catalytically active site of BV-PTP was deleted. Although the vPTPdel mutant was viable in cell culture, it was partially defective in occluded virus production in SF-21 but not TN-368 cell lines. SF-21 cells infected with vPTPdel were heterogeneous in their ability to support occluded virus production. These results suggest that BV-PTP functions in a cell line-specific and possibly a cell cycle-specific fashion. The yield of occlusion bodies, infectivity (concentration of virus causing 50% mortality) and virulence (the time at which 50% of the cells died) of vPTPdel appeared to be normal in insect larvae. We identified a 35-kDa phosphoprotein as a potential target of the BV-PTP in SF-21 cells.

Amino Acid Sequence↗
PubMed1995

Twenty-four-hour mean plasma testosterone concentration declines with age in normal premenopausal women.

The 24-h mean plasma concentration of total testosterone (T) was measured in 33 healthy, regularly cycling, nonobese women between 21 and 51 yr of age. Percent free T was measured in 17 of them. Plasma dehydroepiandrosterone (DHEA) and dehydroepiandrosterone sulfate (DHEAS) were measured in 24 of them, and the DHEA-to-T and DHEAS-to-T ratios were calculated. It was found that the concentration of total T showed a steep decline with age; the regression equation was: T (nanomoles per L) = 37.8 x age-1.12 (r = -0.54; P < 0.003). According to this equation, the expected T concentration of a woman of 40 would be 0.61 nmol/L, about half that of a woman of 21 (1.3 nmol/L). The percent free T did not vary significantly with age, so free T concentration likewise showed a steep decline with age. The DHEA-to-T and DHEAS-to-T ratios were both age invariant, clearly because the levels of DHEA and DHEAS also decline steeply with age, as previously reported.

Adult↗
PubMed1995

Coccidioidomycosis in California: regional outbreak, global diagnostic challenge.

Beginning in 1991, case reports of coccidioidomycosis in California increased dramatically, pursuant to a variety of natural and demographic factors. This highly infectious fungal disease with propensity to disseminate widely, mimic other conditions, and cause pathology at locations distant in place and time is readily treatable if recognized at an early stage. The concentration of military bases in endemic areas and the mobility of military personnel suggest a heightened potential for case presentations elsewhere and a need for elevated diagnostic suspicion on the part of military physicians worldwide. We review three cases of disseminated disease recently referred to our facility.

Adult↗
PubMed1995

Sensitivity to sequential structure in musical savants.

Eight pianists with mild to moderate degrees of mental retardation and 8 comparison subjects were asked to duplicate chord sequences of varying length and musical structure in an immediate memory format. All participants were sensitive to the structural dimensions of the material, and differences between groups were restricted to the least structured material.

Adult↗
PubMed1994

Identification of lef-7: a baculovirus gene affecting late gene expression.

We have examined a 15-kb region of the Autographa californica nuclear polyhedrosis virus (AcMNPV) genome, from 66 to 78 map units, for the presence of genes which transactivate expression from late and very late viral promoters in transient expression assays. One gene in this region activated reporter gene expression approximately six- to eightfold when the reporter gene was under the control of the late promoter of the major capsid protein gene, vp39, or the very late promoter of the polyhedrin gene, polh, but not when the reporter gene was under the control of the early promoter of etl, a homolog of proliferating cell nuclear antigen. The sequence of the predicted polypeptide product of this gene, lef-7, shared no obvious sequence homology to other sequences in available databases. Transcriptional analysis indicated that lef-7 was transcribed early in infection from an initiation site 14 to 16 bp upstream of the putative translational start site and was also transcribed late in infection from an initiation site(s) further upstream. The lef-7 promoter and the promoter of another previously defined late expression factor, lef-3, were both dependent on the multifunctional transregulatory gene, ie-1, for activity in transient expression assays. While sequencing the region of the AcMNPV genome containing lef-7, we also found a 215-codon open reading frame (ORF-215) approximately 1 kb downstream of lef-7 with sequence homology to eIF2 alpha kinases (e.g., rabbit eIF2 alpha and yeast GCN-2 kinase). However, only the six C-terminal conserved domains of protein kinases were present in the predicted ORF-215 product and several of these domains varied from the consensus sequence. ORF-215 did not strongly influence expression from the vp39 promoter-controlled reporter gene in the transient expression assays employed.

Animals↗
PubMed1994

Mutational analysis of a baculovirus major late promoter.

We have used a linker-scan mutation strategy to analyze Pcap99, the proximal promoter of the Autographa californica nuclear polyhedrosis virus (AcMNPV) gene encoding the major capsid protein. A series of recombinant viruses expressing the cat reporter gene under the control of selected mutants of this promoter was constructed. Only mutations that altered bases within a region extending from 8 bp upstream to 6 bp downstream from a TAAG sequence had a significant effect on expression from the late gene promoter. A synthetic promoter consisting of only these 18 bp (Pcapmin) was sufficient to direct late expression. Aside from this small region surrounding the TAAG, no evidence for distinct late activating or repressing sequence elements was obtained. Experiments comparing and combining late and very late gene promoter sequences suggest that late expression is intrinsically determined by the presence and immediate context of a TAAG sequence and that very late expression [as previously shown in Ooi et al., J. Mol. Biol. 210 (1989) 721-736] results from additional modulation of TAAG-dependent expression by downstream promoter elements placed in an appropriate context. A compact combination promoter (95 bp), constructed by fusing Pcapmin to a linker-modified very late polyhedrin promoter, directs strong expression at late and very late times post-infection.

Base Sequence↗
PubMed1994

Identification of three late expression factor genes within the 33.8- to 43.4-map-unit region of Autographa californica nuclear polyhedrosis virus.

A transient transactivation assay system was used in combination with an overlapping Autographa californica nuclear polyhedrosis virus clone library to identify genes involved in late and very late baculovirus gene expression. We have identified three genes within the 33.8- to 43.4-map-unit region of the A. californica nuclear polyhedrosis virus genome which contribute to expression from promoters of the vp39 major capsid protein and polyhedrin genes. One of these three genes corresponds to the previously identified DNA polymerase gene, while the other two genes encode previously unidentified polypeptides of 59,418 and 8,706 Da. None of these genes were required for expression from the early etl promoter.

Animals↗
PubMed1994

Influence of infection route on the infectivity of baculovirus mutants lacking the apoptosis-inhibiting gene p35 and the adjacent gene p94.

The infectivity of Autographa californica nuclear polyhedrosis virus mutants lacking the apoptosis-inhibiting gene p35 is decreased 1,000-fold or more in larvae of the insect Spodoptera frugiperda if the budded form of the virus is administered by hemocoelic injection; this decrease is correlated with the antiviral effects of apoptosis (R. J. Clem and L. K. Miller, J. Virol. 67:3730-3738, 1993). We have extended this correlation by showing that the infectivity of p35 mutant budded virus is restored to wild-type levels by expression of an unrelated baculovirus apoptosis-inhibiting gene, Cp-iap. We have also examined the oral infectivity of the occluded form of mutants lacking p35, the neighboring p94 gene, or both genes by feeding insects occluded virus. The oral infectivity of the p35 mutant was significantly reduced in S. frugiperda larvae, but this reduction (25-fold) was less than that observed for the hemocoelic route of infection (1,000-fold). The disruption of p94 alone had no apparent effect on infectivity by either route. Unexpectedly, however, the disruption of both p35 and p94 restored oral infectivity to nearly wild-type levels but did not exert this compensatory effect on infectivity by hemocoelic injection. Thus, the infectivity of the double p35/p94 mutant is affected in a route-specific manner in S. frugiperda larvae, suggesting a tissue-specific response to p35 and/or p94. Infectivity in a different host, Trichoplusia ni, was unaffected by all the mutants tested, consistent with previous studies indicating a lack of sensitivity to apoptosis in this species. However, T. ni and S. frugiperda larvae infected with p35 mutants failed to exhibit the symptom of morphological disintegration ("melting") typical of a wild-type infection, suggesting that p35 is required for the infection of some tissues in both species.

Animals↗
PubMed1994

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

We have identified a gene required for strong expression of the polyhedrin gene by characterizing a mutant, tsB837, of the baculovirus Autographa californica nuclear polyhedrosis virus (AcMNPV) which is temperature sensitive (ts) for occluded virus production at the nonpermissive temperature. Marker rescue experiments utilizing an overlapping set of AcMNPV genomic clones revealed that the gene responsible for the ts mutant phenotype mapped to a region between 46 and 48 map units. Fragments (2.2 kb) from both wild-type AcMNPV and tsB837 genomes spanning the mutated region were sequenced, and a single nucleotide difference was observed. This mutation is predicted to substitute a single amino acid within a 44.4-kDa polypeptide. Analysis of protein synthesis in wild-type- and mutant-infected cells at the nonpermissive temperature indicated that polyhedrin synthesis was dramatically reduced in the mutant. Northern (RNA) blot analysis revealed that the mutant had markedly reduced levels of polyhedrin transcripts. Transcripts of another very late gene, p10, were also reduced but to a lesser degree. The transcription of two late genes (603 ORF and vp39) was neither reduced nor temporally delayed. Thus, the gene encoding this very late expression factor, designated vlf-1, regulates the levels of very late gene transcripts, and the tsB837 mutation affects the levels of polyhedrin gene transcripts more strongly than those of p10 transcripts. The product of the newly identified gene has a surprising but significant relationship to a family of integrases and resolvases.

Amino Acid Sequence↗
PubMed1994

A mutant baculovirus with a temperature-sensitive IE-1 transregulatory protein.

We have mapped the mutation responsible for the temperature-sensitive (ts) phenotype of tsB821, a mutant of the baculovirus Autographa californica nuclear polyhedrosis virus (H. H. Lee and L. K. Miller, J. Virol. 31:240-252, 1979), to a single nucleotide which changes alanine 432 of the multifunctional regulatory protein IE-1 to a valine. Mapping was done with a combination of marker rescue and transient expression assays, hybrid gene construction by overlap PCR gene splicing, and nucleotide sequence analysis. Cells infected with tsB821 at high multiplicities of infection showed a spectrum of responses from severe cytopathic effects, including apoptosis, to a lack of obvious signs of infection. Protein synthesis in tsB821-infected cells at the restrictive temperature appeared similar to uninfected cell protein synthesis, but viral DNA replication and budded virus production were observed, albeit in a delayed manner. The dependence of early and late promoter activity on the wild-type IE-1 gene, ie-1, was observed in transient expression assays. However, the dependence of early promoter activity on ie-1 was strongest in the absence of other viral genes. Thus, other viral genes appear to be able to compensate, at least in part, for the lack, or low levels, of ie-1 in transient expression assays using early promoters. The mutant should prove useful in further defining the function(s) of IE-1.

Amino Acid Sequence↗
PubMed1994

In vivo and in vitro analyses of recombinant baculoviruses lacking a functional cg30 gene.

The cg30 gene of Autographa californica nuclear polyhedrosis virus (AcMNPV) encodes two sequence motifs, a zinc finger-like motif and a leucine zipper, found in other polypeptides known to be involved in gene regulation. To gain insight into the function of the cg30 product, CG30, we constructed and characterized recombinant viruses lacking a functional cg30 gene. We found that cg30 mutants had no striking phenotype in cell lines derived from Spodoptera frugiperda or Trichoplusia ni or in T. ni larvae. Although cg30 is known to be transcribed as an early monocistronic RNA and as the second cistron of an abundant late bicistronic RNA, production of a CG30-beta-galactosidase fusion protein was observed mainly at early times postinfection. Viruses containing cg30 had a subtle growth advantage over those lacking cg30 after several viral passages in cell culture. We employed transient expression assays to determine whether cg30 and pe-38, an AcMNPV gene that encodes a polypeptide with zinc finger-like and leucine zipper motifs similar to those of cg30, have redundant functions. Although pe-38 may have a role in AcMNPV gene expression, there was no indication that cg30 and pe-38 are functionally redundant.

Alleles↗
PubMed1994

An apoptosis-inhibiting gene from a nuclear polyhedrosis virus encoding a polypeptide with Cys/His sequence motifs.

Two different baculovirus genes are known to be able to block apoptosis triggered upon infection of Spodoptera frugiperda cells with p35 mutants of the insect baculovirus Autographa californica nuclear polyhedrosis virus (AcMNPV):p35 (P35-encoding gene) of AcMNPV (R. J. Clem, M. Fechheimer, and L. K. Miller, Science 254:1388-1390, 1991) and iap (inhibitor of apoptosis gene) of Cydia pomonella granulosis virus (CpGV) (N. E. Crook, R. J. Clem, and L. K. Miller, J. Virol. 67:2168-2174, 1993). Using a genetic complementation assay to identify additional genes which inhibit apoptosis during infection with a p35 mutant, we have isolated a gene from Orgyia pseudotsugata NPV (OpMNPV) that was able to functionally substitute for AcMNPV p35. The nucleotide sequence of this gene, Op-iap, predicted a 30-kDa polypeptide product with approximately 58% amino acid sequence identity to the product of CpGV iap, Cp-IAP. Like Cp-IAP, the predicted product of Op-iap has a carboxy-terminal C3HC4 zinc finger-like motif. In addition, a pair of additional cysteine/histidine motifs were found in the N-terminal regions of both polypeptide sequences. Recombinant p35 mutant viruses carrying either Op-iap or Cp-iap appeared to have a normal phenotype in S. frugiperda cells. Thus, Cp-IAP and Op-IAP appear to be functionally analogous to P35 but are likely to block apoptosis by a different mechanism which may involve direct interaction with DNA.

Amino Acid Sequence↗
PubMed1994

Identification and transcriptional regulation of the baculovirus lef-6 gene.

We identified an Autographa californica nuclear polyhedrosis virus (AcMNPV) gene, the late expression factor 6 gene (lef-6), which is involved in expression from late and very late AcMNPV gene promoters but not from an early AcMNPV gene promoter in transient expression assays. This gene was located within the PstI I fragment of the AcMNPV genome (14.7 to 17.9 map units), immediately downstream of Ac-iap, the AcMNPV homolog of a baculovirus gene family involved in blocking apoptotic programmed cell death. The nature and temporal regulation of both Ac-iap and lef-6 transcripts was examined. Ac-iap and lef-6 were cotranscribed as bicistronic messages at both early and late times postinfection. In addition, lef-6 was transcribed as a monocistronic mRNA by initiation from an early promoter within Ac-iap.

Animals↗
PubMed1994

A baculovirus gene involved in late gene expression predicts a large polypeptide with a conserved motif of RNA polymerases.

We have identified and sequenced a novel baculovirus gene, late expression factor eight gene (lef-8), of Autographa californica nuclear polyhedrosis virus that is necessary for efficient expression from late and very late virus gene promoters in a transient expression assay. The predicted gene product, LEF-8, has a molecular mass of 102 kDa and contains a conserved sequence motif, GXKX4HGQ/NKG, found in DNA-directed RNA polymerases throughout the animal, plant, and microbial kingdoms.

Amino Acid Sequence↗
PubMed1994

Control of programmed cell death by the baculovirus genes p35 and iap.

The SF-21 insect cell line undergoes rapid and widespread apoptosis when treated with actinomycin D or when infected with a mutant of the baculovirus Autographa californica nuclear polyhedrosis virus lacking a p35 gene or a functionally active iap (inhibitor of apoptosis) gene. Here we provide evidence that the basis for the induction of apoptosis by these two different stimuli is the cessation of RNA synthesis. We also show that expression of either p35 or two different functional iap homologs blocks apoptosis independently of other viral genes, indicating that these gene products act directly on the cellular apoptotic pathway. The iap genes encode a C3HC4 (or RING) finger motif found in a number of transcriptional regulatory proteins, as well as two additional Cys/His motifs (baculovirus iap repeats). We show that specific amino acids within both the C3HC4 finger and the N-terminal baculovirus iap repeat are critical for anti-apoptosis function. Overexpression of either mammalian bcl-2 or adenovirus E1B-19K, genes which block apoptosis when overexpressed in a number of mammalian cells, does not block actinomycin D-induced apoptosis in SF-21 cells.

Adenovirus E1B Proteins↗
PubMed1994

Programming for survival: a meeting system that survives 8 years later.

Effective and useful interventions often deteriorate when researchers withdraw their direct supervision. We tested the survival of an intervention designed to produce effective weekly meetings in a student housing cooperative without direct researcher supervision. Chairperson performance, proposals completed per hour, and ratings of chairperson performance all increased when resident staff used a training manual, prompting checklist, and performance reviews. Eight years of follow-up revealed continuing high levels of meeting effectiveness. This study demonstrates a methodology for the direct observation and experimental analysis of intervention survival.

Adult↗
PubMed1993

Expression of hornet genes encoding venom allergen antigen 5 in insects.

Antigen 5, also known as Dol m V, is a major allergen found in the venom of the baldfaced hornet, Dolichovespula maculata. We have inserted the f10 and f17 cDNAs, which encode hornet antigen 5 (HA5) forms 2 (Dol m; VB) and 3 (Dol m VA), respectively, into the genome of the baculovirus, AcMNPV, to produce the recombinant baculovirus gene expression vectors, vEV-HA5f10 and vEV-HA5f17. Insect cells infected with either vEV-HA5f10 or vEV-HA5f17 produce and secrete a novel protein with an electrophoretic mobility which is similar if not identical to authentic mature Dol m V. The gene products also react specifically with a polyclonal antiserum raised to Dol m VB as expected. Dol m V gene products were not acutely toxic when injected into insect larvae. However, infection of fifth instar larvae with vEV-HA5f17 resulted in premature melanization of the larvae and lower weight gain than infection with control virus. Thus, the Dol m V gene product has a subtle, possibly cytotoxic or biochemical effect on insects. The expression systems may prove useful in further structural and functional characterization of these proteins.

Allergens↗