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C J Miller

Publications and source records attributed to C J Miller.

At least 91 records · Page 5Linked to original sources
PubMed1996

Mutational analysis of the role of hydrophobic residues in the 338-348 helix on actin in actomyosin interactions.

Yeast actin mutants with alanines replacing I341 and I345 were studied to assess the role of hydrophobic residues in the alpha-helix 338-348 in interactions with myosin. In structural models of the actomyosin complex, this helix on actin was assigned a prominent role in the strong binding of myosin to actin. Substitution of I341 with alanine reduced the strong binding of actin to myosin subfragment-1 (S1) 9-fold compared to wild-type actin. In addition, the Vmax of the actin-activated S1 ATPase was reduced 4-fold with no change in the Km. In contrast, substitution of I345 with alanine had no significant effect on either the strong binding to S1 or the actin activation of S1 ATPase. The I341A actin filaments were found to slide in the in vitro motility assays at a lower mean velocity (1.6 +/- 0.4 microns/s) than wild-type actin filaments (2.6 +/- 0.3 microns/s). Only 65% of the mutant actin filaments moved in such assays in comparison to 95% of the wild-type filaments. However, addition of 2.0 mM MgADP to the motility assay buffer induced movement of all the I341A filaments at a velocity (1.6 +/- 0.1 microns/s) similar to that of wild-type actin (1.7 +/- 0.1 microns/s). The decrease in motility of the I341A actin filaments in the absence of ADP was attributed to a negative load slowing the mutant filaments and the smaller force produced by the heavy meromyosin and I341A actin system. The latter conclusion was confirmed by showing that a greater percentage of NEM-modified heavy meromyosin (external load) was required for arresting the motion of wild-type actin in the in vitro motility assay than that needed for stopping the I341A filaments.

Actin Cytoskeleton↗
PubMed1996

Myosin-induced changes in F-actin: fluorescence probing of subdomain 2 by dansyl ethylenediamine attached to Gln-41.

Actin labeled at Gln-41 with dansyl ethylenediamine (DED) via transglutaminase reaction was used for monitoring the interaction of myosin subfragment 1 (S1) with the His-40-Gly-42 site in the 38-52 loop on F-actin. Proteolytic digestions of F-actin with subtilisin and trypsin, and acto-S1 ATPase measurements on heat-treated F-actin revealed that the labeling of Gln-41 had a stabilizing effect on subdomain 2 and the actin filaments. DED on Gln-41 had no effect on the values of K(m) and Vmax of the acto-S1 ATPase and the sliding velocities of actin filaments in the in vitro motility assays. This suggests either that S1 does not bind to the 40-42 site on actin or that such binding is not functionally important. The binding of monoclonal antidansyl IgG to DED-F-actin did not affect acto-S1 binding in the absence of nucleotides, indicating that the 40-42 site does not contribute much to rigor acto-S1 binding. Myosin-induced changes in subdomain 2 on actin were manifested through an increase in the fluorescence of DED-F-actin, a decrease in the accessibility of the probe to collisional quenchers, and a partial displacement of antidansyl IgG from actin by S1. It is proposed that these changes in the 38-52 loop on actin originate from S1 binding to other myosin recognition sites on actin.

Actins↗
PubMed1996

Progesterone implants enhance SIV vaginal transmission and early virus load.

Simian immunodeficiency virus (SIV) can cross the intact vaginal epithelium to establish a systemic infection in macaques (mac). Using this SIVmac model, we found that subcutaneous progesterone implants, which could mimic hormonally based contraceptives, thinned the vaginal epithelium and enhanced SIV vaginal transmission 7.7-fold over that observed in macaques treated with placebo implants and exposed to SIV in the follicular phase of the menstrual cycle. Progesterone treatment also increased the number of SIV DNA-positive cells in the vaginal lamina propria as detected by in situ polymerase chain reaction analysis. Moreover, plasma viral RNA was elevated for the first three months in macaques with progesterone implants, and three of the progesterone-treated macaques developed relatively rapid disease courses. This study shows that SIV genital infection and disease course are enhanced by subcutaneous implants containing progesterone when compared with the rate of vaginal transmission in the follicular phase.

Animals↗
PubMed1996

Vaginal transmission of chimeric simian/human immunodeficiency viruses in rhesus macaques.

Chimeric simian/human immunodeficiency viruses (SHIVs) that express the env genes derived from distinct HIV type 1 (HIV-1) isolates were tested for the ability to infect rhesus macaques following intravaginal inoculation. SHIVs containing either the HIV-1 HXBc2 or the HIV-1 89.6 envelope glycoproteins were capable of replicating in intravenously inoculated rhesus macaques. However, intravaginal inoculation of animals with these two SHIVs resulted in infection only with the SHIV containing the HIV-1 89.6 glycoprotein. Thus, properties conferred by the envelope glycoproteins in the chimeric virus affect the ability of particular SHIVs to initiate a systemic infection following vaginal inoculation. These results provide indirect support for the hypothesis that the selection of specific viral variants occurs in the genital tracts of individuals exposed to HIV by sexual contact.

Acquired Immunodeficiency Syndrome↗
PubMed1996

Virus-induced immunosuppression is linked to rapidly fatal disease in infant rhesus macaques infected with simian immunodeficiency virus.

Six newborn rhesus macaques were experimentally infected with pathogenic Simian immunodeficiency virus of macaques (SIVmac251), and three newborn macaques were infected with avirulent SIVmac1A11. The former developed rapidly fatal simian AIDS and died within 26 wk of age, whereas the latter remained clinically normal. Infant monkeys that developed rapidly progressive disease had rapid declines in CD4+ cells and were unable to mount IgG and IgA antibody responses to SIV or to an unrelated antigen, tetanus toxoid. IgM antibody responses were near normal to both SIV-specific and nonspecific antigens. Cytotoxic T lymphocyte (CTL) responses to SIV envelope were observed in animals infected with either virulent or avirulent SIV. These studies demonstrated that virulent SIVmac infection induced a rapid immunosuppression that was both SIV-specific and nonspecific in nature. The observation that virulent strains of SIV can rapidly induce a global immunosuppression provides one explanation for the rapid disease course in some HIV-infected children and supports the strategy of early and vigorous antiviral drug therapy to alter the disease course even if this does not prevent infection.

Amino Acid Sequence↗
PubMed1996

Localization of simian immunodeficiency virus nucleic acid and antigen in brains of fetal macaques inoculated in utero.

Neurological dysfunction has been shown to be associated with human immunodeficiency virus (HIV) infection. The incidence of these abnormalities is greater in HIV-infected children when compared with adults, and the patterns of neurological disease are also known to differ from those observed in the adult population. The reasons for these differences are unclear but are most likely related to the immaturity of the host's immune and central nervous systems at the time of infection. This is thought to be particularly true for infants infected with HIV prenatally. To examine these questions, the brains of fetal rhesus macaques that were infected with SIVmac251 at various time points in utero were examined. Direct fetal inoculations were performed on gestational day (GD) 65 (n = 8; early second trimester), GD 110 (n = 4; early third trimester) and GD 130 (n = 2; mid third trimester), with harvest of fetal tissues on GD 80, 100, 130, or 145. Eleven sham controls were included with harvest at correlative time points. Specimens were examined by routine histology, immunohistochemistry, and in situ hybridization to localize viral antigens and SIV nucleic acid. Histologically, scattered glial nodules, spongiosis, and mineralization were found in the basal ganglia and deep white matter in 4 of the 14 fetuses (3 inoculated on GD 65 and one on GD 110). These fetuses and those without histological lesions had viral nucleic acid and SIV antigen in the stroma of the choroid plexus, meninges, and external granular layer of the cerebellum and in columns of cells in the cortical plate. In contrast to juvenile and adult macaques, very few SIV-positive perivascular mononuclear cells were present. These findings suggest that SIV has a different distribution in the brain of fetal macaques after direct infection when compared with adult or juvenile animals. Furthermore, the results of these studies suggest that differences in neurological disease between pediatric and adult patients with acquired immune deficiency syndrome are most likely related to the time of infection.

Animals↗
PubMed1995

Antiviral cytotoxic T lymphocytes in vaginal mucosa of simian immunodeficiency virus-infected rhesus macaques.

The mucosal immune system of the female reproductive tract is of central importance for protection against sexually transmitted diseases, including HIV; however, this arm of the immune system remains poorly understood. Antiviral CTL responses never have been documented in the genital tract and the role of CTL in this anatomic site is unknown. In this study, CD8+ intraepithelial lymphocytes (IEL) in the vaginas of six simian immunodeficiency virus (SIV)-infected female rhesus macaques were identified by immunohistochemistry to be CD2+ and TCR beta-chain+. In addition, the majority of CD8+ IEL contained TIA-1+ cytoplasmic granules that are associated with CTL activity. CD8+ T cells were isolated from the vaginal epithelium and submucosa and amplified by limiting dilution in the presence of feeder cells. SIV p55gag and/or gp160env-specific lysis was detected in cultures of vaginal epithelial but not submucosal CD8+ T lymphocytes. Estimated SIV-specific precursor CTL frequencies were higher in the vaginal CD8+ IEL population of chronically infected monkeys than in the same cells from acutely infected monkeys or a naive control monkey. These results provide the first demonstration that antiviral CTL are present in the vaginal epithelium, and suggest that a vaccine may be able to generate anti-HIV CTL in the genital mucosa.

Animals↗
PubMed1995

Effects of viral virulence on intrauterine growth in SIV-infected fetal rhesus macaques (Macaca mulatta).

Studies with a simian immunodeficiency virus (SIV)-infected fetal monkey model were conducted with a focus on fetal growth and viral pathogenesis. Twenty-six fetuses were inoculated in utero via ultrasound guidance with an uncloned pathogenic strain of SIV or vehicle during the second or third trimesters [gestational day (GD) 65, 110, or 130], sonographically monitored weekly (biometrics, blood flow), then necropsied at incremental time points postinfection. Peripheral blood hematologic (complete blood counts, clinical chemistries), immunologic (immunophenotyping), and endocrine studies [insulin-like growth factor (IGF), IGF-binding proteins (IGFBP)] were conducted. Severe intrauterine growth restriction (IUGR), oligohydramnios, and altered lymphocyte counts were noted for fetuses infected on GD 65. Less severe effects were detected for fetuses inoculated at the later time points, with severity dependent upon the length of SIV infection in utero. IGF studies indicated significant reductions in IGF-I and elevated immunoreactive levels of IGFBP-3 in infected fetuses during the third trimester. Parallel studies conducted with four fetuses infected on GD 65 with a nonpathogenic, molecularly cloned virus (SIVmac1A11) resulted in normal fetal growth, with no effects on hematopoiesis or IGF/IGFBP levels, and no evidence of clinical disease. Taken together, these studies show that (1) infection of fetuses during the early second trimester with an uncloned pathogenic strain of SIV results in severe IUGR and a disruption in the molar ratio of IGF:IGFBP-3, and (2) outcome of fetal SIV infection is determined by the timing of infection and the virulence of the viral inoculum.

Animals↗
PubMed1995

Isolation of promoters from Brevibacterium flavum strain MJ233C and comparison of their gene expression levels in B. flavum and Escherichia coli.

A promoter probe shuttle vector suitable for the isolation of promoter elements from coryneform bacteria was constructed. This vector carried the neomycin phosphotransferase (NPTII) gene from transposon Tn5 as a reporter gene, and was capable of replication in both Escherichia coli and Brevibacterium flavum. The vector was used in the construction of a B. flavum library of 899 independently isolated promoter clones. Promoters with a wide range of activities in B. flavum, including some very strong promoter elements, were isolated. Comparative analysis suggests that significant differences between B. flavum and E. coli may exist in the determinants of promoter strength.

Base Sequence↗
PubMed1995

Role of charged amino acid pairs in subdomain-1 of actin in interactions with myosin.

Yeast actin mutants with alanines replacing charged amino acid pairs D24/D25, E99/E100, D80/D81, and E83/K84 were studied to assess their role in interactions with myosin. In a previous report Dictyostelium actin filaments with residues D24/D25 or E99/E100 replaced with histidines showed complete or partial loss of filament sliding in the in vitro motility assay [Johara, M., et al. (1993) Proc. Natl. Acad. Sci. U.S.A. 90, 2127-2131]. In the motility experiments reported here, actin filaments with alanines substituted at D24/D25 or E99/E100 moved in the presence of 0.7% methylcellulose at velocities similar to those of wild-type yeast actin. Without methylcellulose, mutant filaments dissociated from the assay surface upon addition of ATP with little or no sliding detected. In contrast to this, filaments with alanines substituted at D80/D81 or E83/K84 were motile in the presence and absence of methylcellulose. Direct binding measurements involving cosedimentation of D24A/D25A and E99A/E100A actins with myosin subfragment-1 (S-1) in the presence of ATP revealed 3- and 2-fold decreases in their binding constants, respectively, compared to wild-type actin. In the absence of ATP all yeast actins had a similar affinity for S-1. A large decrease in the activation of S-1 ATPase was observed for both D24A/D25A and E99A/E100A actins. The D80A/D81A and E83A/K84A actin filaments showed normal S-1 binding and activation of ATPase activity. These results demonstrate the involvement of the D24/D25 and E99/E100 charged residues in the weak binding of myosin to actin and reveal that D80/D81 and E83/K84 residues in the 79-92 helix do not modulate actomyosin interactions.

Actins↗
PubMed1995

Immediate zidovudine treatment protects simian immunodeficiency virus-infected newborn macaques against rapid onset of AIDS.

Simian immunodeficiency virus (SIV) infection of newborn rhesus macaques is a practical animal model of pediatric AIDS. Intravenous inoculation of rhesus newborns with uncloned SIVmac resulted in a high virus load, no antiviral immune responses, severe immunodeficiency, and a high mortality rate within 3 months. In contrast, immediate oral zidovudine (AZT) treatment of SIV-inoculated rhesus newborns either prevented infection or resulted in reduced virus load, enhanced antiviral immune responses, a low frequency of AZT-resistant virus isolates, and delayed disease progression with negligible toxicity. These results suggest that early chronic AZT treatment of human immunodeficiency virus-exposed newborns may have benefits that outweigh its potential side effects.

Administration, Oral↗
PubMed1995

Viral factors determine progression to AIDS in simian immunodeficiency virus-infected newborn rhesus macaques.

To evaluate how viral variants may affect disease progression in human pediatric AIDS, we studied the potential of three simian immunodeficiency virus (SIV) isolates to induce simian AIDS in newborn rhesus macaques. The three virus isolates were previously shown to range from pathogenic (SIVmac251 and SIVmac239) to nonpathogenic (SIVmac1A11) when inoculated intravenously into juvenile and adult rhesus macaques. Six newborn macaques inoculated with pathogenic, uncloned SIVmac251 developed persistent, high levels of cell-associated and cell-free viremia, had no detectable antiviral antibodies, and had poor weight gain; these animals all exhibited severe clinical disease and pathologic lesions diagnostic for simian AIDS and were euthanatized 10 to 26 weeks after inoculation. Two newborns inoculated with pathogenic, molecularly cloned SIVmac239 developed persistent high virus load in peripheral blood, but both animals had normal weight gain and developed antiviral antibodies. One of the SIVmac239-infected neonates exhibited pathologic lesions diagnostic for SAIDS and was euthanatized at 34 weeks after inoculation; the other SIVmac239-infected neonate remained alive and exhibited no significant clinical disease for more than 1 year after inoculation. In contrast, three newborn rhesus macaques inoculated with the nonpathogenic molecular clone, SIVmac1A11, had transient, low-level viremia, seroconverted by 10 weeks after inoculation, had normal weight gain, and remained healthy for over 1 year. These results indicate that (i) newborn rhesus macaques infected with an uncloned, virulent SIVmac isolate have a more rapid, fulminant disease course than do adults inoculated with the same virus, (ii) the most rapid disease progression is associated with lack of a detectable humoral immune response in SIV-infected infant macaques, (iii) a molecularly cloned, attenuated SIV isolate is nonpathogenic in neonatal macaques, and (iv) SIV-infected neonatal macaques exhibit patterns of infection, virus load, and disease progression similar to those observed in human immunodeficiency virus-infected children. This SIV/neonatal rhesus model of pediatric AIDS provides a rapid, sensitive model with which to compare the virulence of SIV isolates and to study the mechanisms underlying the differences in disease progression in human immunodeficiency virus-infected infants.

Animals↗
PubMed1995

Comparing the quality-adjusted life-year output of two treatment arms in a randomized trial.

The quality-adjusted life year (QALY) is an outcome measure with two dimensions: wellness and time. It is difficult to compare the QALY output of two groups when subjects are observed for varying time periods, because both elements of the two-dimensional outcome measure vary in all subjects. This paper evaluates the strengths and weaknesses of five approaches for making these comparisons. These approaches are: a subject-based approach, the elimination of time, calculation of the mean of available data, use of quality-adjusted survival analysis, and calculation of a maximum likelihood estimation. It is concluded that the maximum likelihood estimation offers significant advantages over the other methods.

Female↗
PubMed1995

Actin's view of actomyosin interface.

Actomyosin interactions were examined by using yeast actin mutants with alanines replacing charged amino acid pairs D24/D25, E99/E100, D80/D81, and E83/K84. In the in vitro motility experiments, actin filaments of D24A/D25A or E99A/E100A mutants moved in the presence of 0.7% methylcellulose at the velocities of wild-type actin. Without methylcellulose, these mutant filaments, but not the D80/D81 or E83/K84 filaments, dissociated from the assay surface upon addition of ATP. Measurements of myosin subfragment-1 (S1) binding to D24A/D25A- and E99A/E100A-polymerized actins in the presence of ATP revealed a three- and twofold decrease in their binding constant, respectively, compared with wild-type actin. In contrast to this, all monomeric actins had the same binding affinity for S1. The rates and extents of polymerization of D24A/D25A and E99A/E100A actins by S1 were reduced in comparison to wild-type actin. The local structure of subdomain-2 on actin, as probed by subtilisin cleavage, was not altered for either mutant. A twofold decrease in nucleotide exchange was detected for the D24A/D25A mutant actin. These results demonstrate the involvement of the D24/D25 and E99/E100 residues in the weak binding of myosin to actin and reveal that residues D80/D81 and E83/K84 do not modulate actomyosin interactions.

Actins↗
PubMed1995

Bone marrow monocyte/macrophages are an early cellular target of pathogenic and nonpathogenic isolates of simian immunodeficiency virus (SIVmac) in rhesus macaques.

BACKGROUND: Hematopoietic abnormalities are a common complication of human immunodeficiency virus infection in humans. However, the pathogenesis of these abnormalities remains unclear. Simian immunodeficiency virus (SIV) infection of rhesus macaques is a well-recognized animal model for acquired immunodeficiency syndrome. Our previous studies have determined that in early SIV infection, rhesus macaques develop peripheral blood and bone marrow pathologic changes within the first 14 days after intravenous inoculation. Further investigations were initiated to determine the onset of bone marrow viral infection and the identity of in vivo viral cellular targets in bone marrow during the primary phase of infection in macaques infected with three different strains of SIVmac. EXPERIMENTAL DESIGN: Rhesus macaques experimentally infected with pathogenic uncloned biologic SIVmac, molecularly cloned pathogenic SIVmac-239, or nonpathogenic SIVmac-1A11 were studied at 3, 7, and 14 days postinoculation. Bone marrow samples taken at necropsy were examined to identify early in vivo cellular targets of SIVmac in bone marrow and to correlate hematopathologic lesions with viral infection. In the first 2 weeks after intravenous inoculation, cellular targets of viral infection were identified by a combined in situ hybridization/immunohistochemical technique; changes in bone marrow monocyte/macrophage and CD3+ T lymphocyte populations were evaluated by immunohistochemical techniques. RESULTS: SIV-infected monocyte/macrophages were detected on days 3, 7, and 14 days postinoculation in bone marrow of all monkeys regardless of the viral isolate, whereas only a few SIV-infected CD3+ T lymphocytes were detected in 5 of 18 monkeys. The bone marrow morphologic changes associated with acute SIV infection included macrophage hyperplasia and apparent macrophage activation, diminution of bone marrow T lymphocytes, appearance of lymphoid aggregates, and myeloid and megakaryocytic hyperplasia. CONCLUSIONS: We conclude that bone marrow monocyte/macrophages are an important early cellular target in SIV infection regardless of viral pathogenicity and in vitro cellular tropism. SIV-infected bone marrow monocyte/macrophages may play a key role in the pathogenesis of bone marrow lesions and further dissemination and persistence of virus infection.

Animals↗