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Biomedical subjects

M R White

Publications and source records attributed to M R White.

At least 19 recordsLinked to original sources
PubMed2006

Influenza a viruses upregulate neutrophil toll-like receptor 2 expression and function.

Neutrophils are involved in the initial host response to influenza A virus (IAV) infection and exhibit both activation and depressed function after exposure to the virus. We demonstrate that IAV causes rapid upregulation of Toll-like receptor 2 (TLR2) expression on neutrophils. The neutrophil agonists, formyl-methylpleucyl-alanine (fMLP), C5a and lipopolysaccharide did not alter neutrophil TLR2 expression, whereas PMA and the microbial TLR2 ligands, peptidoglycan (PGN) and zymosan, reduced it. To determine the functional significance of IAV-induced increase in TLR2 expression, IAV-treated neutrophils were exposed to PGN, Staphylococcus aureus (S. aureus) and zymosan. Pretreatment with IAV resulted in significantly increased uptake of S. aureus and zymosan and accelerated neutrophil apoptosis when combined with S. aureus. IAV-treated cells generated significantly more H(2)O(2) in response to PGN. These results indicate that IAV increases neutrophil surface expression of TLR2 and modulates functional responses to ligands that bind TLR2. These findings may clarify IAV-induced perturbation of neutrophil functions in vivo.

Complement C5a↗
PubMed2004

Primitive neuroectodermal neoplasia in coho salmon, Oncorhynchus kisutch.

Twelve coho salmon, approximately 8 weeks old, were each observed to have a single neoplasm involving the dorsolateral axial skeletal musculature. The neoplasm was closely associated with the vertebrae in all cases. The neoplasm was composed of islands containing small cells with round and occasional spindeloid morphology. Neoplastic cells had basophilic cytoplasm and vesicular nuclei. These cells exhibited immuno-positivity only for vimentin and S-100 protein. Ultrastructurally, neoplastic cells had nuclei with a predominance of euchromatin, cytoplasm containing marked amounts of rough endoplasmic reticulum, scant amounts of smooth endoplasmic reticulum, and scattered mitochondria. Rudimentary cell junctions were occasionally observed between adjacent neoplastic cells. Based on the close association of these neoplasms with the vertebrae as well as the histologic, ultrastructural, and immunohistochemical findings, these neoplasms were considered to all be primitive neuroectodermal neoplasms.

Animals↗
PubMed2002

Evaluation of nested polymerase chain reaction, pepsin/trypsin digest, and histology for the detection of Myxobolus cerebralis in salmonid fishes of Indiana and Michigan.

The objectives of this study were to survey fish from state hatcheries in Indiana and Michigan and to compare the nested polymerase chain reaction (PCR) test with pepsin/trypsin digest (PTD) and histopathology for the diagnosis of whirling disease (WD). One group of 40 and 9 groups of 60 fish heads, for a total of 580 samples, were submitted from hatcheries in Indiana and Michigan. These samples were examined for myxozoan spores using histopathology, PTD, and PCR tests. The heads were hemisectioned, and one half was fixed in 10% neutral-buffered formalin for histopathologic examination. The other half was processed for PTD. Some of the sediment was examined for the presence of myxozoan spores, and the rest was prepared for the nested PCR. Histologic examinations did not reveal Myxobolus cerebralis in any of the 580 samples. One hundred serial step sections, taken at 5-microm intervals, were evaluated for samples with positive spore identification by PTD. Histologic examination of these sections failed to reveal any myxozoan parasites. Myxozoan spores were observed in 16.9% (98/580) of samples in sediment after PTD. Spores morphologically similar to those of M. cerebralis were observed in 1.0% of PTD samples (n = 6). The nested PCR indicated that M. cerebralis spores were present in 0.5% of samples (n = 3). All 3 nested PCR-positive samples came from the same hatchery, however, spores of M. cerebralis were seen in 1 sample, spores of other myxozoan species were seen in the second sample, and spores were not seen in the third sample. When comparing the PTD to the nested PCR test, the PTD diagnosed 1 true positive, 5 false positives, 2 false negatives, and 572 true negatives, for a sensitivity of 33% and a specificity of 99.1%. Screening for M. cerebrallis infection in this study indicated a low prevalence of the disease. Histopathology was a very insensitive indicator of WD. The PCR test was highly specific and was used to differentiate spores of M. cerebralis from similar spores of other species.

Animals↗
PubMed2001

Hyperadrenocorticism associated with excessive sex hormone production by an adrenocortical tumor in two dogs.

An 11-year-old spayed female Labrador Retriever and a 9-year-old castrated male miniature Poodle were evaluated because of clinical signs of hyperadrenocorticism. Cortisol testing did not support a diagnosis of hypercortisolemia in either dog; however, imaging studies revealed unilateral adrenal tumors in both dogs. Serum concentrations of 17-hydroxyprogesterone, progesterone, and estradiol were high in both dogs, and androstenedione concentrations were also high in 1 dog. It is suspected that sex hormone secretion by the adrenal tumors in these dogs resulted in clinical signs of hyperadrenocorticism. Clinical signs and hormonal abnormalities resolved in the male dog after surgical resection of the tumor. There was no improvement in clinical signs after treatment with mitotane in the female dog, which died 2 months after diagnosis. Histologic evaluation confirmed the presence of adrenocortical carcinoma in both dogs.

Adrenal Cortex Neoplasms↗
PubMed2001

Increased antiviral and opsonic activity of a highly multimerized collectin chimera.

Altering the carbohydrate binding properties of surfactant protein D (SP-D) [e.g., by replacing its carbohydrate recognition domain (CRD) with that of either mannose binding lectin (MBL) or conglutinin] can increase its activity against influenza A virus (IAV). The current study demonstrates that the degree of multimerization of SP-D is another independent determinant of antiviral activity. A chimeric collectin containing the N-terminus and collagen domain of human SP-D and the CRD of MBL formed high-molecular-weight multimers similar to those previously described for human SP-D. Using several complementary assays, and diverse viral strains, the chimeric multimers showed greater anti-IAV activity than similarly multimerized preparations of SP-D or incompletely oligomerized preparations of the chimera. More highly multimerized preparations of the chimera also caused greater increases in uptake of IAV by neutrophils. These studies may have implications for development of collectins as therapeutic agents and understanding of natural variations in susceptibility to IAV infection.

Animals↗
PubMed2001

Transcription inhibition induced by modified triple helix-forming oligonucleotides: a quantitative assay for evaluation in cells.

Oligonucleotides can bind to double-stranded DNA in a sequence-specific manner to form triple helices. Uniformly modified, pyrimidine-rich oligodeoxyribonuclotides containing internucleosidic N3'-P5' phosphoramidate linkages are known to form very stable triplexes with their DNA target. Psoralen-conjugated triple helix-forming oligonucleotides (Pso-TFOs) can additionally be photo-induced to become irreversibly bound to their targeted DNA sequence. Here, we have examined the ability of various 15-mer phosphoramidate TFOs targeted to the HIV-1 polypurine tract (PPT) sequence to prevent transcription elongation in cell cultures; the PPT sequence has been cloned in the transcribed region of a reporter firefly luciferase gene (luc) and transient expression experiments performed. We show that the level of transcription inhibition of the reporter gene in cells perfectly correlates with the amount of covalent triplex at the PPT site. The efficacy of non-covalent triplexes (either omitting the irradiation step with the psoralen conjugate, or using the unsubstituted oligonucleotide) has been studied in our expression system; the oligonucleotides were introduced into living cells by cationic lipid-mediated delivery or directly into the cell nucleus by microinjection. This experimental approach allowed us to evaluate the intrinsic activity of triplexes as transcriptional inhibitors; transcription elongation was inhibited in cells in a sequence-dependent and concentration-dependent manner. This experimental system is convenient for quantitative and fast evaluation of new chemistries of antigene oligonucleotides as inhibitors of gene expression in cells and in vivo.

Base Sequence↗
PubMed2001

Phosphoramidate oligonucleotides as potent antisense molecules in cells and in vivo.

Antisense oligonucleotides are designed to specifically hybridize to a target messenger RNA (mRNA) and interfere with the synthesis of the encoded protein. Uniformly modified oligonucleotides containing N3'-P5' phosphoramidate linkages exhibit (NP) extremely high-affinity binding to single-stranded RNA, do not induce RNase H activity, and are resistant to cellular nucleases. In the present work, we demonstrate that phosphoramidate oligonucleotides are effective at inhibiting gene expression at the mRNA level, by binding to their complementary target present in the 5'-untranslated region. Their mechanism of action was demonstrated by comparative analysis of three expression systems that differ only by the composition of the oligonucleotide target sequence (HIV-1 polypurine tract or PPT sequence) present just upstream from the AUG codon of the firefly luciferase reporter gene: the experiments have been done on isolated cells using oligonucleotide delivery mediated by cationic molecules or streptolysin O (SLO), and in vivo by oligonucleotide electrotransfer to skeletal muscle. In our experimental system phosphoramidate oligonucleotides act as potent and specific antisense agents by steric blocking of translation initiation; they may prove useful to modulate RNA metabolism while maintaining RNA integrity.

5' Untranslated Regions↗
PubMed2001

Photoperiodic regulation of prolactin gene expression in the Syrian hamster by a pars tuberalis-derived factor.

Syrian hamsters exhibit a marked seasonal variation in prolactin secretion. The aim of this study was to analyse the nature of the photoperiodic regulation of prolactin gene expression, and to define the role of melatonin and the pars tuberalis of the anterior pituitary in this process. Pituitary prolactin gene expression, restricted to the pars distalis, was increased in hamsters maintained in long daylengths (16 h : 8 h, light : dark) compared to hamsters exposed to short daylengths (8 h : 16 h, light : dark) for 8-12 weeks. Analysis of single cells by in situ hybridization showed that photoperiod had no effect on the percentage of pars distalis cells expressing prolactin mRNA, but shifted the frequency distribution of prolactin mRNA expression per cell, such that in long photoperiods a greater proportion of cells were recruited to a higher expressing population. In vitro coculture of hamster pars tuberalis fragments increased prolactin promoter-driven luciferase activity in stably transfected GH3 cells in a dose- and duration-dependent manner. Conditioned medium from hamster and ovine pars tuberalis also activated the prolactin promoter. Furthermore, basal and forskolin-stimulated conditioned medium from hamster pars tuberalis increased prolactin mRNA expression in primary cultures of pars distalis cells. Melatonin attenuated the activity of pars tuberalis-conditioned medium but had no direct effect on either prolactin mRNA expression or secretion in pars distalis cell cultures. Finally, pars tuberalis fragments from long photoperiod hamsters stimulated prolactin gene promoter activity to a greater extent than those from short photoperiod hamsters. In conclusion, this study provides the first evidence in a seasonal mammal that the synthesis of prolactin depends on photoperiodic modulation of a pars tuberalis-derived factor. Our data support further the hypothesis that seasonal modulation of prolactin gene expression depends upon a melatonin-dependent paracrine action of the pars tuberalis on pars distalis lactotrophic cells.

Animals↗
PubMed2001

Characterization of the ethanol-inducible alc gene-expression system in Arabidopsis thaliana.

Controlled expression of transgenes in plants is key to the characterization of gene function and the regulated manipulation of growth and development. The alc gene-expression system, derived from the filamentous fungus Aspergillus nidulans, has previously been used successfully in both tobacco and potato, and has potential for use in agriculture. Its value to fundamental research is largely dependent on its utility in Arabidopsis thaliana. We have undertaken a detailed function analysis of the alc regulon in A. thaliana. By linking the alcA promoter to beta-glucuronidase (GUS), luciferase (LUC) and green fluorescent protein (GFP) genes, we demonstrate that alcR-mediated expression occurs throughout the plant in a highly responsive manner. Induction occurs within one hour and is dose-dependent, with negligible activity in the absence of the exogenous inducer for soil-grown plants. Direct application of ethanol or exposure of whole plants to ethanol vapour are equally effective means of induction. Maximal expression using soil-grown plants occurred after 5 days of induction. In the majority of transgenics, expression is tightly regulated and reversible. We describe optimal strategies for utilizing the alc system in A. thaliana.

Arabidopsis↗
PubMed2001

Persistent synchronized oscillations in prolactin gene promoter activity in living pituitary cells.

PRL gene expression in the anterior pituitary gland responds rapidly to different hormonal signals. We have investigated the long-term timing of transcriptional activation from the PRL, GH, and cytomegalovirus promoters in response to different stimulus duration, using real-time imaging of luciferase expression in living stably transfected GH3 cells. Long-term stimulation of serum-starved cells with 50% serum induced a homogeneous rise in PRL promoter activity, with subsequent heterogeneous fluctuations in luciferase activity in individual cells. When cells were subjected to a 2-h pulse of 50% serum, followed by serum-free medium, there were long-term (approximately 50 h) synchronized, homogeneous oscillations in PRL promoter activity. This response was PRL-specific, because in GH3 cells expressing luciferase from the GH or cytomegalovirus promoters, a serum pulse elicited no oscillations in luciferase expression after an initial transient response to serum. The PRL promoter may therefore be a template for an unstable transcription complex subject to stochastic regulation, allowing an oscillatory transcriptional response to physiological signals. This suggests that precise timing and coordination of cell responses to different signal-duration may represent a novel mechanism for coordinating long-term dynamic changes in transcription in cell populations.

Blood Physiological Phenomena↗
PubMed2001

BCL-2 family expression in human neutrophils during delayed and accelerated apoptosis.

The human neutrophil spontaneously undergoes apoptosis, but this type of cell death can be delayed or accelerated by a wide variety of agents. There are wide discrepancies in the literature regarding the expression of the Bcl-2 family of proteins in human neutrophils. Here, we show that A1, Mcl-1, Bcl-X(L), and Bad are major transcripts in human neutrophils and that levels of these transcripts are cytokine regulated. However, no Bcl-X(L) protein was detected in Western blots. Protein levels for the proapoptotic proteins Bad, Bax, Bak, and Bik remained constant during culture, despite changes in the levels of mRNA for these gene products. These proapoptotic proteins were extremely stable, having very long half-lives. In contrast, A1 and Mcl-1 transcripts were extremely unstable (with approximately 3-h half-lives), and Mcl-1 protein was also subject to rapid turnover. These results indicate that neutrophil survival is regulated by the inducible expression of the short-lived Mcl-1 and possibly the A1 gene products. In the absence of their continued expression, these prosurvival gene products are rapidly turned over, and then the activity of the stable death proteins predominates and promotes apoptosis.

Adult↗
PubMed2000

Mechanism of binding of surfactant protein D to influenza A viruses: importance of binding to haemagglutinin to antiviral activity.

Collectins are important in the initial containment of a variety of pathogens, including influenza A virus (IAV). We provide the first systematic evaluation of the oligosaccharide-binding sites for pulmonary surfactant protein D (SP-D) on specific IAV coat glycoproteins and define the relationship between this binding and antiviral activity. With the use of several techniques, SP-D was found to bind via its carbohydrate-recognition domain (CRD) to mannosylated, N-linked carbohydrates on the HA(1) domain of the haemagglutinin (HA) and on the neuraminidase of IAV. Using a set of IAV strains that differed in the level and site of glycosylation, and a panel of recombinant collectins, we found that binding of SP-D to the globular domain of the HA was critical in mediating the inhibition of viral haemagglutination activity and infectivity. We also demonstrated that the pattern of binding of a collectin to IAV glycoproteins can be modified by altering the monosaccharide-binding affinity of its CRD or by linking the CRD to a different N-terminal/collagen domain. These studies clarify the mechanisms of viral neutralization by collectins and might be useful in engineering collectins for enhanced antiviral activity.

Amidohydrolases↗
PubMed2000

Apoptosis is rapidly triggered by antisense depletion of MCL-1 in differentiating U937 cells.

Mcl-1 is a member of the Bcl-2 protein family, which has been shown to delay apoptosis in transfection and/or overexpression experiments. As yet no gene knockout mice have been engineered, and so there is little evidence to show that loss of Mcl-1 expression is sufficient to trigger apoptosis. U937 cells constitutively express the antiapoptotic protein Bcl-2; but during differentiation, in response to the phorbol ester PMA (phorbol 12 beta-myristate 13 alpha-acetate), Mcl-1 is transiently induced. The purpose of this investigation was to determine the functional role played by Mcl-1 in this differentiation program. Mcl-1 expression was specifically disrupted by chimeric methylphosphonate/phosphodiester antisense oligodeoxynucleotides to just 5% of control levels. The depletion of Mcl-1 messenger RNA (mRNA) and protein was both rapid and specific, as indicated by the use of control oligodeoxynucleotides and analysis of the expression of other BCL2 family members and PMA-induced tumor necrosis factor-alpha (TNF-alpha). Specific depletion of Mcl-1 mRNA and protein, in the absence of changes in cellular levels of Bcl-2, results in a rapid entry into apoptosis. Levels of the proapoptotic protein Bax remained unchanged during differentiation, while Bak expression doubled within 24 hours. Apoptosis was detected within 4 hours of Mcl-1 antisense treatment by a variety of parameters including a novel live cell imaging technique allowing correlation of antisense treatment and apoptosis in individual cells. The induction of Mcl-1 is required to prevent apoptosis during differentiation of U937 cells, and the constitutive expression of Bcl-2 is unable to compensate for the loss of Mcl-1. (Blood. 2000;96:1756-1763)

Apoptosis↗
PubMed2000

Enhanced antiviral and opsonic activity of a human mannose-binding lectin and surfactant protein D chimera.

The carbohydrate recognition domains (CRDs) of human serum mannose-binding lectin (MBL) and pulmonary surfactant protein D (SP-D) have distinctive monosaccharide-binding properties, and their N-terminal and collagen domains have very different quaternary structures. We produced a chimeric protein containing the N terminus and collagen domain of human SP-D and the neck region and CRD of human MBL (SP-D/MBLneck+CRD) to create a novel human collectin. The chimera bound to influenza A virus (IAV), inhibited IAV hemagglutination activity and infectivity, and induced aggregation of viral particles to a much greater extent than MBL. Furthermore, SP-D/MBLneck+CRD caused much greater increases in neutrophil uptake of, and respiratory burst responses to, IAV than MBL. These results indicate that pathogen interactions mediated by the MBL CRD are strongly influenced by the N-terminal and collagen-domain backbone to which it is attached. The presence of the CRD of MBL in the chimera resulted in altered monosaccharide binding properties compared with SP-D. As a result, the chimera caused greater aggregation and neutralization of IAV than SP-D. Distinctive functional properties of collectin collagenous domains and CRDs can be exploited to generate novel human collectins with potential for therapy of influenza.

Animals↗
PubMed2000

In vivo localisation and stability of human Mcl-1 using green fluorescent protein (GFP) fusion proteins.

Mcl-1 is an anti-apoptotic member of the Bcl-2 family of proteins. We have expressed full length and mutated GFP:Mcl-1 fusion proteins to define structural motifs that control protein localisation and stability. When expressed in U-937 cells, full length Mcl-1 locates primarily within mitochondria and its half-life was approximately 3 h, which was identical to the native, endogenously expressed protein. When the terminal 20 amino acids from the C-terminus of the protein were detected, the protein was diffused in the cytoplasm, but its stability was unaffected. This confirms that this region is responsible for efficient targeting to mitochondria. Surprisingly, deletion of 104 amino acids (residues 79-183) that contain putative PEST sequences and other stability regulating motifs, did not affect protein stability.

Amino Acid Motifs↗
PubMed2000

Interaction in vivo and in vitro of the metastasis-inducing S100 protein, S100A4 (p9Ka) with S100A1.

The calcium-binding protein S100A4 (p9Ka) has been shown to cause a metastatic phenotype in rodent mammary tumor cells and in transgenic mouse model systems. mRNA for S100A4 (p9Ka) is present at a generally higher level in breast carcinoma than in benign breast tumor specimens, and the presence of immunocytochemically detected S100A4 correlates strongly with a poor prognosis for breast cancer patients. Recombinant S100A4 (p9Ka) has been reported to interact in vitro with cytoskeletal components and to form oligomers, particularly homodimers in vitro. Using the yeast two-hybrid system, a strong interaction between S100A4 (p9Ka) and another S100 protein, S100A1, was detected. Site-directed mutagenesis of conserved amino acid residues involved in the dimerization of S100 proteins abolished the interactions. The interaction between S100A4 and S100A1 was also observed in vitro using affinity column chromatography and gel overlay techniques. Both S100A1 and S100A4 can occur in the same cultured mammary cells, suggesting that in cells containing both proteins, S100A1 might modulate the metastasis-inducing capability of S100A4.

Animals↗
PubMed2000

Targeted inhibition of transcription elongation in cells mediated by triplex-forming oligonucleotides.

Triple-helix-forming oligonucleotides (TFOs) bind in the major groove of double-stranded DNA at oligopyrimidine small middle dotoligopurine sequences and therefore are candidate molecules for artificial gene regulation, in vitro and in vivo. We recently have described oligonucleotide analogues containing N3'-P5' phosphoramidate (np) linkages that exhibited efficient inhibition of transcription elongation in vitro. In the present work we provide conclusive evidence that np-modified TFOs targeted to the HIV-1 polypurine tract (PPT) sequence can inhibit transcriptional elongation in cells, either in transient or stable expression systems. The same constructs were used in transient expression assays (target sequence on transfected plasmid) and in the generation of stable cell lines (target sequence integrated into cellular chromosomes). In both cases the only distinguishable feature between the cellular systems is the presence of an insert containing the wild-type PPT/HIV-1 sequence, a mutated version with two mismatches, or the absence of the insert altogether. The inhibitory action induced by np-TFOs was restricted to the cellular systems containing the complementary wild-type PPT/HIV-1 target, and consequently can be attributed only to a triple-helix-mediated mechanism. As a part of this study we also have applied an imaging technique to quantitatively investigate the dynamics of TFO-mediated specific gene silencing in single cells.

Animals↗