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Biomedical subjects

M W White

Publications and source records attributed to M W White.

At least 19 recordsLinked to original sources
PubMed1995

Lymphocyte p56L32 is a RNA/DNA-binding protein which interacts with conserved elements of the murine L32 ribosomal protein mRNA.

In previous studies of the ribosomal protein L32 mRNA, we demonstrated that a conserved polypyrimidine tract found in the 5'-untranslated region (5'-UTR) was required for translational regulation in vivo and that a 56-kDa protein (p56L32) from T-lymphocytes specifically interacts with this sequence [Kaspar, R. L., Kakegawa, T., Cranston, H., Morris, D. R. & White, M. W. (1992) J. Biol. Chem. 267, 508-514]. Here we show that p56L32 binding to the L32 5'-UTR is complex and requires other 5'-UTR RNA sequences in conjunction with the polypyrimidine tract. Deletion and site-directed mutagenesis studies revealed that binding of p56L32 to the L32 5'-UTR requires a second RNA element, GGUGGCUGCC, 15 nucleotides downstream from the polypyrimidine tract. In contrast, L32 RNA transcripts altered in this downstream element were good substrates for binding of the polypyrimidine binding proteins from HeLa nuclear extracts, indicating that these proteins have RNA-binding specificities distinct from p56L32. Competition analysis demonstrated that p56L32 will bind to DNA as well as RNA with identical sequence specificity and similar affinity. Single or double-stranded DNAs composed of the L32 5'-UTR sequences were found to specifically compete with L32 RNA transcripts for p56L32 binding. The L32 5'-UTR downstream element, GGUGGCUGCC, which is required for p56L32 binding, has previously been implicated as a transcriptional element of the L32 gene. The ability of p56L32 to bind this sequence as DNA or RNA suggests p56L32 may have a dual role in the regulation of ribosomal protein mRNA accumulation and translation.

Animals↗
PubMed1995

Identification of Eimeria bovis merozoite cDNAs using differential mRNA display.

Differences in gene expression between Eimeria bovis sporozoites and first-generation merozoites were analyzed using the technique of differential mRNA display. Approx. 5% of the sequences detected in first-generation merozoites appear to be unique relative to sporozoites. Several of the bands corresponding to merozoite-specific gene expression were isolated and cloned. Northern blot analysis revealed that the cDNA fragments DMZ-7, DMZ-8 and NMZ-6 hybridized to mRNAs expressed at > 50-fold higher levels in merozoites relative to sporozoites. A fourth cDNA fragment, NMZ-4, hybridized to a mRNA expressed at 3-fold higher levels in merozoites. Further characterization demonstrated that expression of DMZ-8 in E. bovis-infected bovine cells begins as early as 12 h after sporozoite invasion and continues throughout the entire 14 days of first-generation schizogony. Sequence analysis of each of the four merozoite cDNAs failed to identify any significant similarity to any entries in the GenBank database, suggesting that these developmentally regulated genes may be unique to coccidian parasites.

Animals↗
PubMed1995

Sporozoites of Toxoplasma gondii lack dense-granule protein GRA3 and form a unique parasitophorous vacuole.

The invasion of host cells by sporozoites of Toxoplasma gondii leads to the formation of parasitophorous vacuoles that are distinctly different from those surrounding tachyzoites. In sporozoite-infected cells, the fluid-filled space surrounding the sporozoite is many times larger in volume than the sporozoite, essentially lacks granular or tubular structures, and has no detectable continuous parasitophorous vacuolar membrane when prepared by conventional electron microscopic methods. Consistent with the ultrastructural differences, dense-granule protein GRA3, which associates with the parasitophorous vacuolar membrane of tachyzoites, was not detected by indirect immunofluorescence in sporozoite-infected cells 2-12 h post-inoculation or by Western blot analysis of sporozoite extracts. Western blots incubated with the alpha ROP/DG antiserum, which recognizes tachyzoite rhoptry and dense-granule proteins, revealed numerous other antigenic differences between sporozoites and tachyzoites. Cell cultures inoculated with sporozoites were monitored at various intervals for the expression of GRA3 and the developmentally-regulated tachyzoite surface protein SAG1. Expression of SAG1 and GRA3 was first observed in 30% of the sporozoite-infected cells at 12 and 15 h post-inoculation, respectively, and in all intracellular parasites at 24 h. Parasite replication was only observed in sporozoite-infected cells that were positive for GRA3 and SAG1. Thus, these data indicate that sporozoites and their interaction with host cells differ substantially from tachyzoites and the expression of tachyzoite-specific proteins is likely required for parasite replication.

AIDS-Related Opportunistic Infections↗
PubMed1995

Plant bacterial spores, active systemically as a separate entity, play a significant role in human illnesses such as cancer, granulomas, AIDS, and milky white abdominal ascites that currently defies recognition.

There is currently an unrecognized chapter in medical illnesses, occurring in living human beings, that defies recognition in explaining the diseases' origin and growth, and failing accurately to account for the pathophysiology involved. It is pertinent, therefore, to alert medical science, based upon facts as uncovered by my research studies (1-5), that plant bacterial spores can exist as a separate entity systemically. Being totally devoid of the presence of their adult origin, they can survive with a degree of viability. Ultimately, they may become embedded within, or nearby, a network of cells, consisting of the reticuloendothelial, squamous, or epithelial group. With an ensuing adequate but compatible circulating flow of blood by the host to the specific tissue site as muscles, bone, or bone marrow, it can thus lead to the various pathophysiological changes and illnesses that currently defy an accountable recognition.

Abdomen↗
PubMed1995

An improved method for isolating RNA from coccidian oocysts.

A method, based on the disruption of eimerian oocysts in a French pressure cell in the presence of guanidine isothiocyanate, has been developed to isolate large quantities of high quality total RNA efficiently. This procedure results in a 12.5-fold greater number of oocysts broken, and a 22-fold greater yield of total RNA than from disruption by conventional grinding in liquid N2. In addition, the RNA isolated by the French pressure cell method was of equal or superior quality when compared to RNA isolated by grinding. This procedure provides a significant improvement in RNA extraction from eimerian oocysts and will greatly facilitate the study of gene expression in this important group of parasites.

Animals↗
PubMed1994

Developmental regulation of an Eimeria bovis mRNA encoding refractile body-associated proteins.

Eimeria bovis antigens defined by the monoclonal antibody (mAb) 2.4 are associated with the refractile bodies of sporozoites and are found in the parasitophorous vacuole and host cell cytoplasm during schizogony. Screening of an E. bovis oocyst cDNA library with mAb 2.4 resulted in the identification of a single unique cDNA sequence (Eb-25/50). Comparison of the predicted protein sequence of Eb-25/50 revealed a high degree of identity to an Eimeria tenella refractile body protein and mAb 2.4 was found to cross-react with refractile bodies from Eimeria acervulina, demonstrating that these proteins are highly conserved among eimerian species. Measurements of Eb-25/50 mRNA showed that the multiple proteins recognized by mAb 2.4 are encoded by a single mRNA species whose kinetics of expression during sporulation and schizogony closely correlated with protein expression. Consistent with multiple Eb-25/50 proteins arising from a single polypeptide, results from a Southern analysis of E. bovis genomic DNA indicated that Eb-25/50 mRNA is derived from a single copy gene. The presence of Eb-25/50 proteins in the host cytoplasm during schizogony, the high degree of conservation of these proteins, and the apparent complex post-translational modification raises interesting questions about the biochemistry of these proteins during eimerian development.

Amino Acid Sequence↗
PubMed1994

A specific oxidant is the prime factor in cancer cells' origin and growth.

Sufficient evidence is present to conclude the fact that there is a specific anion acceptor (oxidant) substance actively functioning within the living malignant cell. It is this oxidant that possesses the physiological properties to accept the negative charged electrons while releasing or expelling the oxygen molecules. This oxidant, as studies indicate, consists of a specific delicate chemical entity. It arises from one of several varieties of plant like bacterial microorganisms, whose spores, once formed, have the genetic capability of embedding within a cell or sac, lie quiescently, while retaining, and still holding viable its electron acceptor chemical complex. These spores are identified in the group of microorganisms known as ascospores. It is important to emphasize that it is the spore forms only and not the adult form in any manner that enters the animal cell. It is this oxidant present within the latter spores that will become activated when there develops an adequate circulating flow of blood to provide the transportation of the metabolites, electrons, cations, CO2 and oxygen molecules to and from the involved cells. The ultimate response, with this oxidant's presence is the striking physiological alteration whereby respiration and metabolism is henceforth basically anaerobic. There is likewise the ability too for the spores under these circumstances to reproduce asexually to produce further spores by the method of abstriction from the blastospore's embryonic phase.

Anaerobiosis↗
PubMed1993

Developmental gene expression in Eimeria bovis.

By differential screening of stage-specific cDNA libraries of Eimeria bovis, we have identified and isolated a large set of genes that are regulated during development of the sporozoites and merozoites. Duplicate lifts of cDNA libraries constructed from partially sporulated oocysts and merozoites were probed with radioactively labeled first-strand cDNA prepared from partially sporulated oocyst and merozoite mRNA. Out of 60,000 plaques screened in each case, over 250 plaques from the partially sporulated oocyst library preferentially hybridized with the oocyst cDNA probe and 67 plaques from the merozoite library preferentially hybridized with the merozoite cDNA probe. Three of the oocyst phage and 7 of the merozoite phage were selected for further characterization. Northern analysis revealed a common pattern of mRNA expression for the oocyst cDNA clones. Consistent with the results of the differential screen, no hybridization to merozoite RNA was detected with any of these 3 oocyst cDNA clones. The expression of the merozoite cDNA clones was more complex, with 3 different classes of merozoite genes being identified based on their pattern of developmental regulation. Although each of the merozoite clones was expressed to some extent during sporulation, in all cases, expression was higher in merozoites than in partially sporulated oocysts, consistent with the restriction of expression defined by the differential screen. Sequence analysis revealed that 2 of the merozoite cDNA clones encode elongation factor 1 alpha and the ubiquitin/ribosomal protein fusion, and 1 of the sporozoite cDNAs displays a significant identity to insulin-degrading enzyme. The developmental expression of E. bovis genes involved in protein synthesis and degradation provides additional evidence for the importance of regulation of protein metabolism during parasite development.

Amino Acid Sequence↗
PubMed1992

A regulatory cis element and a specific binding factor involved in the mitogenic control of murine ribosomal protein L32 translation.

The mRNA encoding ribosomal protein L32 redistributes from untranslated subribosomal particles into polysomes after mitogenic activation of quiescent T-lymphocytes and fibroblasts. To identify the regions of the L32 mRNA which are important in regulating its cytoplasmic location we constructed a plasmid containing the murine L32 cDNA under the control of the Rous sarcoma virus (RSV) long terminal repeat promoter and introduced this construct into murine 3T3 fibroblasts. The mRNA transcribed from the RSV-L32 construct redistributed from subribosomal particles into polysomes in response to mitogenic activation in a manner similar to endogenous L32 mRNA. A conserved polypyrimidine region present at the 5' terminus of all ribosomal protein mRNAs is required for translational regulation of L32 mRNA since deletion of this sequence resulted in a mRNA that was not sequestered in subribosomal particles in quiescent cells. A radioactive RNA probe containing the first 34 nucleotides of the L32 5'-untranslated region, including the polypyrimidine region, specifically interacted with a protein of about 56 kDa. This protein did not bind detectably to RNA probes lacking the polypyrimidine sequence. Binding activity was similar in protein extracts made from resting and activated cells, suggesting that binding of the 56-kDa protein as measured in this assay is not regulated. This protein is a member of what may be an emerging family of polyribopyrimidine-binding proteins with diverse biochemical functions.

3T3 Cells↗
PubMed1992

Metabolism of the malignant cell, in vivo, is anaerobic and significantly plays a factor in the pathway to carcinogenesis.

The malignant cell, in vivo, metabolizes and respires anaerobically. For a good many years scientists have been aware of anaerobia being present in malignant growths. The prevailing opinion is that the malignant cell is able to remain viable in an anaerobic medium. This assumption is incorrect and is actually misleading. My investigative studies indicates that the malignant cell metabolizes and respires intracellularly so that the consequence of such a physiology is anaerobic and the basis for such anaerobia is a metabolic phenomenon. There is a reason for this anaerobiosis. Studies demonstrated the fact that there are present within the malignant cell and in the immediate area bacterial spores arising from one of several varieties of plant bacteria. And with an adequate circulating flow of blood, by the animal (human) host, to that particular site, provides the metabolites, enzymes, and the exchange of gases to enhance this abnormal physiopathological anaerobiosis. It is this intracellular anaerobic metabolism within an animal cell that becomes the basis for its malignant transformation.

Anaerobiosis↗
PubMed1992

Metabolism of the malignant cell, the role of bacterial spores, and a pictorial presentation to substantiate the latter's presence as an etiological factor in carcinogenesis.

The respiration and metabolism of the malignant cell, while in vivo, is anaerobic. This is contrary to a normal animal (human) cell, whose respiration and metabolism is aerobic. There is a factor within the malignant cell which produces a reducing or 'deoxygenating' phenomenon, creating the metabolic anaerobiosis. An animal cell cannot survive without the ultimate oxygen molecule. Yet the malignant cell continues to grow and enlarge despite this oxygen absence. Even more surprising is the fact that the malignant cell requires the circulating flow of blood, otherwise that deficient site or area will ulcerate, slough, or necrotize. Investigative studies clearly indicate that there are present 'spores' of a specific group type of plant micro-organism within the malignant cell, and in the immediate and circumferential area to account for this abnormal physiology. In summarizing all the experimentally uncovered facts, it becomes apparent that the malignant cell, while in vivo, is the consequence of 3 factors of productivity. For the first factor there is the requirement of a tissue site whereby there is an accumulate number of the chronic defense cells as the reticuloendothelial group, squamous, and/or epithelial cells. The second factor requires the presence and phagocytosis of these specific plant bacterial spores that can survive genetically within a sac or cell, and third, for the adequate circulating flow of blood by the host. Because of this, 3 factor product activity, Koch's postulate cannot be fulfilled as a criterion for the etiology of the cancer cell. We have, however, a pictorial summary of the pertinent uncovered facts that, when added together, presents a credible, logical, and valid conclusion to support the concept that these specific bacterial spores contribute to the pathway of activity to associate them as the malefactor in carcinogenesis.

Anaerobiosis↗
PubMed1992

A nucleic acid-based test for detection of Fasciola hepatica.

The use of nucleic acid techniques in the diagnosis of parasitic infection has become increasingly widespread. An oligonucleotide probe derived from a rRNA sequence was developed for the detection of Fasciola hepatica in its intermediate snail host Pseudosuccinea columella. Total RNA obtained from whole adult liver flukes was used in a polymerase chain reaction to isolate and amplify a region of approximately 650 base pairs in the small subunit rRNA. This portion of the ribosomal cDNA, which contains highly conserved regions as well as variable regions, was subcloned and sequenced. In comparison to known small subunit rRNA sequences, a sequence unique to F. hepatica was identified and an oligonucleotide probe (CS4) for detection of F. hepatica was developed. A northern blot analysis using CS4 successfully identified small subunit rRNA from F. hepatica. Slot-blot analysis determined that RNA derived from 5 miracidia can be detected with CS4. Moreover, a slot blot utilizing CS4 distinguished RNA derived from snails infected with F. hepatica from RNA of uninfected snails.

Animals↗
PubMed1991

Activation of dense human tonsilar B cells. Induction of c-myc gene expression via two distinct signal transduction pathways.

Antibodies to surface Ig or to the B cell marker CD20 trigger resting human B cells in similar yet distinct ways. Either antibody induces five-fold increases in the expression of the protooncogene, c-myc, as detected with semi-quantitative Northern blot assays. The induction of c-myc mRNA by anti-IgM or anti-CD20 is blocked by inhibitors of protein kinase C (PKC) such as staurosporine and by pretreatment of B cells with phorbol esters to reduce cellular PKC levels. This suggests that PKC is involved in the pathways stimulated by both anti-IgM and anti-CD20. However, anti-CD20, unlike anti-IgM, does not activate significant increases in inositol triphosphate or intracellular-free calcium. Further, anti-CD20-triggered elevation of c-myc mRNA is inhibited by pertussis and cholera toxins, whereas the pathway initiated by anti-IgM if anything is stimulated by pertussis toxin and unchanged by cholera toxin. Further differences in the nature of these two signals were seen when the expression of adhesion/recognition molecules were examined. Anti-IgM consistently induces increased expression of the adhesion molecules CD54 (I-CAM-1) and B7/BB-1 on B cells, but anti-CD20 does not. Yet both anti-CD20 and anti-IgM increase class II MHC, CD18 (LFA-1 beta-chain) and LFA-3 levels. These data suggest that the way in which B cells are activated may influence their surface phenotype and possibly subsequent migration or cell-cell interactions.

Antibodies, Anti-Idiotypic↗
PubMed1991

Pathway to carcinogenesis: the role of bacterial spores.

There are bacteria present within the malignant cell, but they are present only as spores. Although their origin stems from a branching or budding plant type of microorganism, the latter is not physically present and is not involved, per se, in the malignant cell's etiology. It is the primitive but now the asexual reproductive, conidial unicellular, ovular, or spherical shaped bodies, arising from the adult plant microorganism, in consequence to a duressing environment, but also the ones with the genetic capability of surviving within a sac or cell, that are present within the malignant cell. It is these spores, reproducing as spores within the malignant cell and within the immediate surrounding area, as long as there exists an adequate circulating flow of blood by the animal (human) host, to account for the anaerobic or preferably 'deoxygenating' metabolism that occurs in the malignant cell and is the reason for the cellular accumulation and tumor growth. This type of metabolism, respiration, and reproductive pattern is similar to that of the plant type of microorganism activity. In comparison, the healthy non-tumor tissue of animal (human) cells are genetically and primarily aerobic in respiration and metabolism. There are substantial suggestive findings and structural facts uncovered experimentally to definitely establish credence to the presence of those spores within the malignant cell and to align their presence to the pathway of activity, resulting in the etiology and physiopathology of the malignant cell and growth.

Animals↗