Biomedical subjects

L K Miller

Publications and source records attributed to L K Miller.

At least 19 recordsLinked to original sources

PubMed2003

Sex difference in the effect of obesity on 24-hour mean serum gonadotropin levels.

To determine the effect of obesity on serum gonadotropin levels and any possible sex difference in the effect, we measured the 24-hour mean serum follicle-stimulating hormone (FSH) and luteinizing hormone (LH) concentrations in 62 healthy men with Body Mass Index (BMI) ranging from 20 - 94 and 61 healthy, regularly cycling women with BMIs ranging from 19 - 76. We also measured free testosterone (T) and estradiol (E2) in these subjects. There was a significant negative correlation between serum FSH and BMI in men: FSH(IU/L) = 49.9 x BMI -0.567; r = - 0.376, p = 0.0026; but a significant positive correlation between serum FSH and BMI in women: FSH(IU/L) =7.66 +/- 0.071 x BMI; r = 0.302, p = 0.018. Serum LH was weight-invariant in both sexes. In men, free T was negatively correlated with BMI: Free T (nmol/L) = 0.74 - 0.0068 x BMI; r = 0.585, p = 0.0381; and free E2 was positively correlated with BMI: Free E2 (pmol/L) = - 1.03 +/- 0.057 x BMI; r = 0.50, p = 0.0014. In obese women as a group, free T was higher than in lean women (33 +/- 6.8 S.E.M. vs. 17.4 +/- 2.0 pmol/L; p < 0.0001), and free E2 was also higher than in lean women: (6.90 +/- 0.80 vs. 4.84 +/- 0.55 pmol/L; p = 0.046). Of the many cases of hypothalamic-pituitary hormonal dysregulation that have been reported in obesity, none has been studied for sex differences. Our results mandate that possible sex differences be investigated in all cases of dysregulation.

Animals↗

PubMed2001

Deletions in the Ac-iap1 gene of the baculovirus AcMNPV occur spontaneously during serial passage and confer a cell line-specific replication advantage.

The PstI-I region of the Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV) genome was previously shown to be a frequent target of spontaneous deletions during serial virus passage in TN-368 cells (Kumar and Miller, Virus Res. 7 (1987) 335). Analysis of two of these serial passage mutants showed that a portion of the Ac-iap1 gene was deleted. To directly test the effect of loss of Ac-iap1, three different deletions in Ac-iap1 were introduced into recombinant viruses and the ability of these viruses to replicate was examined in two cell lines, TN-368 and SF-21, as well as in two species of insect larvae, Trichoplusia ni and Spodoptera frugiperda. The mutant viruses were indistinguishable from wild type or control revertant virus in their ability to infect larvae of either species. Moreover, no effect was seen on the rate of replication or the overall amounts of budded or occluded virus produced in cultured cells. However, in co-infection experiments using TN-368 cells, it was consistently observed that mutants lacking a functional Ac-iap1 gene out-competed control viruses carrying Ac-iap1. Interestingly, this replication advantage was only evident in the TN-368 cell line, the cell line used for the original serial passage experiments, and not in SF-21 cells.

Animals↗

PubMed2001

Phase I study of paclitaxel given by seven-week continuous infusion concurrent with radiation therapy for locally advanced squamous cell carcinoma of the head and neck.

PURPOSE: Paclitaxel is one of the most active agents for squamous cell carcinoma of the head and neck (SCCHN) and an in vitro radiosensitizer. The dose-response relationship for paclitaxel may depend more on exposure duration than on peak concentration. This National Cancer Institute-sponsored phase I trial was designed to determine the feasibility of combining continuous-infusion (CI) paclitaxel with concurrent radiation therapy (RT). PATIENTS AND METHODS: Patients with previously untreated stage IVA/B SCCHN were eligible. Primary end points were determination of the maximum-tolerated dose, dose-limiting toxicity, and pharmacokinetics for paclitaxel given by CI (24 hours a day, 7 days a week for 7 weeks) during RT (70 Gy/7 weeks). RESULTS: Twenty-seven patients were enrolled and assessable for toxicity. Nineteen of the patients who completed > or = 70 Gy were assessable for response. Grade 3 skin and mucosal acute reactions occurred at 10.5 mg/m(2)/d, but uninterrupted treatment was possible in five of six patients. At 17 mg/m(2)/d, skin toxicity required a 2-week treatment break for all three patients. The mean paclitaxel serum concentration at dose levels > or = 6.5 mg/m(2)/d exceeded that reported to achieve in vitro radiosensitization. Initial locoregional control was achieved in 14 (58%) of 24 of patients treated to 70 Gy, and control persisted in nine (38%). CONCLUSION: CI paclitaxel with concurrent RT is a feasible and tolerable regimen for patients with advanced SCCHN and good performance status. Preliminary response and survival data are encouraging and suggest that further study is indicated. The recommended phase II dose of paclitaxel by CI is 10.5 mg/m(2)/d with RT for SCCHN.

Adult↗

PubMed2001

Organization of the neurons of origin of the descending pathways from the ferret superior colliculus.

The superior colliculus (SC), through its descending projections to the brainstem and spinal cord, is involved in initiating sensory-driven orienting behaviors. Ferrets are carnivores that hunt both above and below ground using visual (and auditory) cues in the daylight but non-visual cues in darkness and in subterranean environments. The present investigation sought to determine whether the ferret SC shows organizational features similar to those found in other visually dominant animals (e.g. cats), or whether characteristics of colliculi from non-visually dominant animals (e.g. rodents) prevail. Injection of retrograde tracer into the identified targets of the colliculus (cervical spinal cord, the contralateral pontomedullary reticular formation, or the ipsilateral pontine reticular formation) labeled tectospinal, crossed tectoreticular, and ipsilateral tectoreticular neurons, respectively, within the adult ferret SC. Labeled tectospinal and crossed tectoreticular neurons were far outnumbered by neurons with ipsilateral reticular projections. Like those of their visually dominant relatives, ferret tectospinal neurons were well represented throughout the anterior-posterior extent of the SC and crossed tectoreticular neurons tended to be distributed more broadly across the intermediate gray layer than those of rodents. Thus, even though ferrets perform well as subterranean predators where non-visual cues initiate orienting behaviors, these anatomical characteristics indicate that their colliculi are organized similar to that of their visually dominant, carnivorous relatives.

Animals↗

PubMed2001

The changing economics of HIV care.

New advances in human immunodeficiency virus (HIV) monitoring and therapeutics have led to dramatic changes in the course of HIV disease. We evaluated our closed clinic of 425 HIV patients over the period 1995-1998 to determine the cost effectiveness of these changes in care. We found that the costs of antiretroviral therapy tripled over the period of observation, but that these increases were largely offset by major declines in inpatient and home health expenditures. In addition, we found that annual mortality among our HIV patients had declined by 90%. We calculated that the cost per life-year gained is about $17,500, which compares favorably with medical expenditures for renal dialysis or advanced cardiac disease.

Antiviral Agents↗

PubMed2001

Terminal deoxynucleotidyl transferase-positive cells in human tonsils.

To study the possible cellular origin of recently recognized indolent terminal deoxynucleotidyl transferase (TdT)-positive T-lymphoblastic proliferations of the tonsils and oropharynx, we studied normal human tonsils for the presence of TdT-positive cells. TdT-positive cells were readily demonstrated in the tonsils from 15 children and adults by immunohistochemical staining. TdT-positive cells were distributed in discrete foci at the periphery of lobules of lymphoid tissue and adjacent to fibrous septa and had the morphologic features of small to medium-sized lymphocytes. Double-antibody staining indicated the TdT-positive cells had the phenotype of uncommitted early lymphoid precursors (CD3-, CD79a-, CD10-). Foci of TdT-positive cells were not identified in 6 reactive lymph nodes studied as controls. These studies indicate that tonsils, like bone marrow and thymus, are sites of lymphopoiesis. The presence of TdT-positive precursor cells in human tonsils may be a factor in the pathogenesis of recently described indolent T-lymphoblastic proliferations involving the tonsils and oropharynx. The presence of TdT-positive cells in human tonsils should not be misinterpreted as evidence of lymphoblastic lymphoma or leukemia.

Adolescent↗

PubMed1999

An exegesis of IAPs: salvation and surprises from BIR motifs.

The BIR (baculovirus IAP repeat) motif is a conserved sequence of approximately 70 amino acids that was identified originally in the 'inhibitor of apoptosis' (IAP) family of proteins. BIR-containing proteins (BIRPs) are found in viruses, yeast and metazoans. Recent genetic analysis of a nematode BIRP demonstrated an essential role in cytokinesis instead of apoptosis. It is likely that BIRs originated in eukaryotes to serve a role in cytokinesis and/or mitotic spindle function during cell division and that, with gene duplication, the more recent adaptation of some BIRPs to the regulation of apoptosis was possible. IAPs interact with a variety of proteins, including members of the caspase protease family. This article discusses current research on the structure and function of the BIR motifs and how it could provide insight into the function of BIRPs in cell division.

Animals↗

PubMed1999

The savant syndrome: intellectual impairment and exceptional skill.

Occasionally, people with developmental disability display skills at a level inconsistent with their general intellectual functioning, so-called "savant" behavior. Studies of savant behavior are reviewed to determine their relevance to notions about the importance of general intellective functions in the development of exceptional skill. It is concluded that (a) the skill exhibited by savants shares many characteristics with that in people without disability, (b) the skill is usually accompanied by normative levels of performance on at least some subtests of standardized measures of cognitive achievement, and (c) it is unclear whether savants have distinctive cognitive strengths or motivational dispositions, though their relative prevalence among people with certain kinds of disability suggests predisposing constraints. The author proposes that these skills typically reflect highly elaborated preconceptual representational systems.

Achievement↗

PubMed1999

Baculovirus p33 binds human p53 and enhances p53-mediated apoptosis.

In vertebrates, p53 participates in numerous biological processes including cell cycle regulation, apoptosis, differentiation, and oncogenic transformation. When insect SF-21 cells were infected with a recombinant of the baculovirus Autographa californica nuclear polyhedrosis virus (AcMNPV) overexpressing human p53, p53 formed a stable complex with the product of the AcMNPV orf92, a novel protein p33. The interaction between p53 and p33 was further confirmed by immunoprecipitation studies. When individually expressed in SF-21 cells, human p53 localized mainly in the nucleus whereas baculovirus p33 displayed diffuse cytoplasmic staining and punctuate nuclear staining. However, coexpression of p33 with p53 resulted in exclusive nuclear localization of p33. In both SF-21 and TN-368 cells, p53 expression induced typical features of apoptosis including nuclear condensation and fragmentation, oligonucleosomal ladder formation, cell surface blebbing, and apoptotic body formation. Coexpression of p53 with a baculovirus inhibitor of apoptosis, p35, OpIAP, or CpIAP, blocked apoptosis, whereas coexpression with p33 enhanced p53-mediated apoptosis approximately twofold. Expression of p53 in SF-21 cells stably expressing OpIAP inhibited cell growth in the presence or absence of p33. Thus, human p53 can influence both insect cell growth and death and baculovirus p33 can modulate the death-inducing effects of p53.

Amino Acid Sequence↗

PubMed1999

In vivo and in vitro analysis of baculovirus ie-2 mutants.

Upon transient expression in cell culture, the ie-2 gene of Autographa californica nuclear polyhedrosis virus (AcMNPV) displays three functions: trans activation of viral promoters, direct or indirect stimulation of virus origin-specific DNA replication, and arrest of the cell cycle. The ability of IE2 to trans stimulate DNA replication and coupled late gene expression is observed in a cell line derived from Spodoptera frugiperda but not in a cell line derived from Trichoplusia ni. This finding suggested that IE-2 may exert cell line-specific or host-specific effects. To examine the role of ie-2 in the context of infection and its possible influence on the host range, we constructed recombinants of AcMNPV containing deletions of different functional regions within ie-2 and characterized them in cell lines and larvae of S. frugiperda and T. ni. The ie-2 mutant viruses exhibited delays in viral DNA synthesis, late gene expression, budded virus production, and occlusion body formation in SF-21 cells but not in TN-5B1-4 cells. In TN-5B1-4 cells, the ie-2 mutants produced more budded virus and fewer occlusion bodies but the infection proceeded without delay. Examination of the effects of ie-2 and the respective mutants on immediate-early viral promoters in transient expression assays revealed striking differences in the relative levels of expression and differences in responses to ie-2 and its mutant forms in different cell lines. In T. ni and S. frugiperda larvae, the infectivities of the occluded form of ie-2 mutant viruses by the normal oral route of infection was 100- and 1,000-fold lower, respectively, than that of wild-type AcMNPV. The reduction in oral infectivity was traced to the absence of virions within the occlusion bodies. The infectivity of the budded form of ie-2 mutants by hemocoelic injection was similar to that of wild-type virus in both species. Thus, ie-2 mutants are viable but exhibit cell line-specific effects on temporal regulation of the infection process. Due to its effect on virion occlusion, mutants of IE-2 were essentially noninfectious by the normal route of infection in both species tested. However, since budded viruses exhibited normal infectivity upon hemocoelic injection, we conclude that ie-2 does not affect host range per se. The possibility that IE-2 exerts tissue-specific effects has not been ruled out.

Animals↗

PubMed1999

Activation of baculovirus very late promoters by interaction with very late factor 1.

Very late factor 1 (VLF-1) of Autographa californica multicapsid nuclear polyhedrosis virus (AcMNPV) activates the transcription of two genes, polyhedrin (polh) and p10, during the final, occlusion-specific phase of infection. Using transient expression assays responsive to VLF-1, we identified linker scan mutations in the polh and p10 promoters which abolished or weakened the ability of the promoters to respond to stimulation by VLF-1. These mutations were located between the transcriptional and translational initiation sites, a region previously shown to be essential for the burst of expression during the very late phase. Addition of partially purified, epitope-tagged VLF-1 to DNA encompassing this "burst sequence" resulted in a shift in the gel electrophoretic mobility of the DNA, indicating that VLF-1 forms a complex with DNA. Addition of an antibody specific for the epitope tag of VLF-1 decreased the mobility of the DNA further, confirming the presence of VLF-1 in the complex. DNase I footprint assays revealed that VLF-1 partially purified from either insect cells or bacterial cells interacted with the burst sequences of both the polh and p10 very-late promoters. Linker scan mutations within the burst sequences severely impaired interaction between VLF-1 and the promoters. We propose that VLF-1 transactivates the polh and p10 promoters by interacting with the burst sequences.

Animals↗

PubMed1999

The baculovirus PE38 protein augments apoptosis induced by transactivator IE1.

While studying apoptosis induced by baculovirus transactivator IE1 in SF-21 cells, we found that the levels of IE1-induced apoptosis were increased approximately twofold upon cotransfection with the baculovirus early pe38 gene. However, no apoptotic activity was observed in cells transfected with pe38 alone, even when placed under the control of a constitutive promoter. Thus, pe38 was able to augment IE1-induced apoptosis but was unable to induce apoptosis when expressed in SF-21 cells alone. PE38, the full-length product of pe38, is a nuclear protein with RING finger and leucine zipper motifs. Deletion of the amino-terminal region, which contains a putative nuclear localization motif, resulted in cytoplasmic localization of the PE38 mutants. These N-terminal deletion mutants were unable to enhance IE1-induced apoptosis. Mutation of a single conserved leucine (L242) of the leucine zipper motif also eliminated the ability of PE38 to augment apoptosis induced by IE1. In contrast, PE38 mutants with alanine substitutions for conserved cysteine residues (C109 or C138) of the RING finger motif were able to increase IE1-induced apoptosis to levels equivalent to those of wild-type PE38. We propose that PE38 is one of at least two viral factors which collectively evoke a cellular apoptotic response during baculovirus infection.

Amino Acid Sequence↗

PubMed1998

A mutational analysis of the baculovirus inhibitor of apoptosis Op-IAP.

A family of antiapoptotic regulators known as inhibitors of apoptosis (IAPs) was initially identified and functionally described in baculoviruses, and IAP homologues are now known in insects, birds, and mammals. Baculovirus and Drosophila IAPs inhibit apoptosis induced by Drosophila proapoptotic proteins Reaper, HID, and GRIM and physically interact with them through their baculovirus IAP repeat (BIR) region. Here we examined the functional importance of BIR and RING finger motifs of Orgyia pseudotsugata nuclear polyhedrosis virus Op-IAP and D-IAP1 in binding to and inhibiting HID. In the absence of both the BIR1 and RING motifs, the BIR2 regions of Op-IAP and D-IAP1 were able to associate with HID and block HID-induced apoptosis. Mutation of conserved amino acid residues within the BIR and RING finger motifs revealed that the conserved residues within BIR2 were essential for Op-IAP to inhibit apoptosis. However, most of the conserved residues of the BIR2 were not required for HID binding. A region at the carboxy-proximal end of BIR2 was essential for the association of Op-IAP with HID. Thus binding to HID is necessary but not sufficient to block HID-induced apoptosis: the conserved residues within BIR2 must have an additional role in blocking apoptosis. These findings demonstrate that the region encompassing a single BIR of Op-IAP and D-IAP1 can be sufficient for physical interaction with and inhibition of apoptosis induced by HID.

Amino Acid Sequence↗

PubMed1998

The Drosophila inhibitor of apoptosis D-IAP1 suppresses cell death induced by the caspase drICE.

Many members of the Inhibitor of Apoptosis (IAP) family inhibit cell death and existing data suggest at least two mechanisms of action. Drosophila IAPs (D-IAP1 and D-IAP2) and a baculovirus-derived IAP, Op-IAP, physically interact with and inhibit the anti-apoptotic activity of Reaper, HID, and Grim, three genetically defined inducers of apoptosis in Drosophila, while human IAPs, c-IAP1, c-IAP2, and X-IAP interact with a number of different proteins including specific members of the caspase family of cysteine proteases which are crucial in the execution of cell death. We have examined whether insect-active IAPs can inhibit apoptosis induced by selected caspases, Drosophila drICE, Sf-caspase-1, and mammalian caspase-3, in insect SF-21 cells. D-IAP1 inhibited apoptosis induced by the active forms of all three caspases tested and physically interacted with the active, but not the proform of drICE. MIHA, the mouse homolog of X-IAP and an effective inhibitor of caspase-3, also interacted with and blocked apoptosis induced by active drICE but was relatively ineffective in blocking Sf-caspase-1. Op-IAP and D-IAP2 were unable to inhibit effectively any of the active caspases tested and failed to interact with drICE. The Drosophila IAPs and Op-IAP, but not MIHA, blocked HID-initiated activation of pro-drICE. We conclude that D-IAP1 is capable of inhibiting the activation of drICE as well as inhibiting apoptosis induced by the active form of drICE. In contrast, D-IAP2 and Op-IAP are more limited in their inhibitory targets and may be limited to inhibiting the activation of caspases.

Animals↗

PubMed1998

Control of baculovirus polyhedrin gene expression by very late factor 1.

vlf-1 is a baculovirus gene that regulates very late gene expression (J. R. McLachlin and L. K. Miller, J. Virol., 68, 7746-7756, 1994) and also plays a crucial role in the replication of the budded form of Autographa californica nuclear polyhedrosis virus (AcMNPV) (S. Yang and L. K. Miller, "Expression and mutational analysis of the baculovirus very late factor 1 (vlf-1) gene." Virology, 245, 99-109, 1998). To examine the influence of vlf-1 expression on baculovirus infection, we constructed recombinant viruses that expressed only low levels of VLF-1 and recombinants with vlf-1 under the control of different promoters. Viruses with mutant alleles of vlf-1 that produced low levels of VLF-1 replicated the budded form of the virus normally but produced no occlusion bodies. Thus, a higher concentration of VLF-1 was needed to activate very late gene expression than was needed to support budded virus production. By altering the level and/or timing of vlf-1 expression, the timing of polyhedrin gene (polh) expression, which normally occurs very late in infection, could be advanced or delayed. Early overexpression of vlf-1 increased the level of expression from the polh promoter but caused premature cellular disintegration. The data indicate that VLF-1 is the limiting factor in very late gene expression and that the level of VLF-1 controls the onset of occlusion.

Animals↗

PubMed1998

A baculovirus mutant defective in PKIP, a protein which interacts with a virus-encoded protein kinase.

We have found that a temperature-sensitive mutant of the baculovirus AcMNPV, tsB97, is defective in PKIP, the product of ORF24 which was previously found to interact with and stimulate the activity of a virus-encoded protein kinase, PK-1. The mutant lacks the ability to form plaques and occlusion bodies at the nonpermissive temperature. The mutant displays several properties which suggest a defect in the latter half of the late phase of infection; these properties include a delay in the shutoff of host protein synthesis, the presence of aberrant electron-dense bodies associated with the virogenic stroma, and the production of few, if any, progeny budded virus. A study of the expression of selected late genes showed no difference in the timing or level of transcription or translation of most late genes. However, elevated levels of the late 6.9K protein, a protamine-like protein, were observed in mutant-infected cells at 24 h postinfection, suggesting a defect in the regulation of this protein. Two polypeptides, 40 and 6 kDa, exhibited considerably higher levels of steady-state phosphorylation in wt-infected cells versus tsB97-infected cells at 24 h p.i. and could be candidates for PK-1/PKIP-mediated phosphorylation. The tsB97 mutant also displayed a severe defect in very late gene transcription which accounts for its inability to form occlusion bodies. The effect of PKIP on very late gene transcription may be a secondary effect of the block in the late phase of infection. PKIP showed no ability to transactivate expression from a very late promoter in transient expression assays.

Animals↗

PubMed1998

Expression and mutational analysis of the baculovirus very late factor 1 (vlf-1) gene.

We have examined the expression and function of a gene, vlf-1, of Autographa californica nuclear polyhedrosis virus that is known to encode a regulator of very late gene transcription. Western blot analysis revealed that vlf-1 is expressed during the late phase of infection, primarily from 15 to 24 h postinfection. VLF-1 localized in the cell nucleus and was also present in the nucleocapsids of virus particles. Mapping of vlf-1 mRNA by primer extension showed that transcription initiates at a TAAG motif 71 bp upstream of the vlf-1 open reading frame. Disruption of this TAAG motif abolished the ability of vlf-1 to stimulate transcription from the very late polyhedrin gene (polh) promoter in transient expression assays, suggesting that vlf-1 expression is controlled by the TAAG motif. Using a highly efficient system to construct recombinant viruses with modifications in vlf-1, we confirmed that the TAAG motif was essential. Furthermore, efforts to construct null mutants of vlf-1 failed, suggesting that vlf-1 is an essential gene for virus replication. Computer-assisted sequence homology searches place vlf-1 in the lambda phage integrase family (McLachlin and Miller, 1994). None of the strictly conserved residues of this family which are found in vlf-1 could be changed in the viral genome, implying that the putative integrase activity of VLF-1 is associated with the essential function of vlf-1. However, mutation of a crucial active-site tyrosine did not affect the ability of vlf-1 to transactivate the polh promoter in transient expression assays, indicating that the very late transcriptional activity of VLF-1 does not require the integrase activity.

Animals↗

PubMed1998

Mutational analysis of Caenorhabditis elegans CED-4.

Much of our knowledge concerning the genetics that regulate cell death has come from the studies of cell death during the development of the nematode Caenorhabditis elegans. Of the 14 genes identified as components of nematode cell death pathways, two genes, ced-3 and ced-4, are required to promote cell death and a third, ced-9, blocks cell death. Recent studies show CED-4 to be an activator of CED-3 and CED-9 to be an inhibitor of CED-4. Two published sequence alignments suggest that CED-4 contains a death effector domain (DED), a protein sequence motif present in other death signaling proteins like Fadd and Flice; one study suggests a DED sequence similarity near the N-terminus while the other found sequence similarity near the C-terminus of CED-4. Using mutational analysis we have tested the functional significance of the conserved residues found within the putative DEDs of CED-4. Mutations in two conserved residues within the putative N-terminal DED of CED-4 affected its function, while mutations in the conserved residues within the putative C-terminal DED had no effect on CED-4 function. Our results do not support the presence of a DED in the C-terminus of CED-4 and suggest a potential role for the N-terminus in CED-4 function, possibly as a DED or as a CARD (caspase recruitment domain). We also found that CED-9 associated with all the CED-4 mutants and inhibited the activity of all the active-CED-4 mutants.

Amino Acid Sequence↗