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Biomedical subjects

T Douglas

Publications and source records attributed to T Douglas.

At least 19 recordsLinked to original sources
PubMed2005

Interactions of anti-sigma factor antagonists of Mycobacterium tuberculosis in the yeast two-hybrid system.

Anti-sigma factor antagonists (anti-anti-sigma factors) play critical roles in regulating the expression of alternative sigma factors in response to specific stress signals. The Clusters of Orthologous Groups (COG) database has identified the existence of six genes, Rv0516c, Rv1364c, Rv1365c, Rv1904, Rv2638 and Rv3687c (grouped under the cluster COG1366), encoding potential anti-sigma factor antagonists in Mycobacterium tuberculosis. These molecules are speculated to regulate the expression of sigma factor SigF of M. tuberculosis in response to stress signals. Since signaling occurs via physical interactions of proteins (protein-protein interaction), we investigated whether the anti-sigma factor antagonists of M. tuberculosis interact with anti-sigma factor RsbW (Rv3287c) or the sigma factor SigF (Rv3286c) in the yeast two-hybrid system. The results revealed that most of the anti-sigma factor antagonists interact with either RsbW or SigF or both. In addition, some anti-sigma factor antagonists also displayed limited interactions between themselves. These interactions suggest that they possibly transduce some signals to SigF and between themselves.

Bacterial Proteins↗
PubMed2005

Biomimetic synthesis of a H2 catalyst using a protein cage architecture.

A biomimetic approach has been used to develop an artificial hydrogenase that catalyses the efficient reduction of protons producing hydrogen gas. Analogous to the unique biological metal clusters found in hydrogenase enzymes, the engineered active sites are small, well-defined Pt clusters deposited on the interior of a heat shock protein cage architecture with stoichiometries of 150 to 1000 Pt per protein cage. The proton reduction reaction is driven by visible light through a coupled reaction with Ru(bpy)3(2+) and methyl viologen as an electron-transfer mediator. Hydrogen production rates are comparable to those of hydrogenase on a per protein basis and exceed production rates of other reported Pt-based catalysts. These results demonstrate the utility of a biomimetic approach toward addressing the needs of hydrogen production.

Archaeal Proteins↗
PubMed2004

Effects of culturing on the population structure of a hyperthermophilic virus.

The existence of a culturing bias has long been known when sampling organisms from the environment. This bias underestimates microbial diversity and does not accurately reflect the most ecologically relevant species. Until now no study has examined the effects of culture bias on viral populations. We have employed culture-independent methods to assess the diversity of Sulfolobus spindle-shaped viruses (SSVs) from extremely hyperthermal environments. This diversity is then compared to the viral diversity of cultured samples. We detected a clear culturing bias between environmental samples and cultured isolates. This is the first study identifying a culture bias in a viral population.

Archaeal Viruses↗
PubMed2004

Noninvasive monitoring of stem cell transfer for muscle disorders.

In this study the ability of magnetodendrimers to efficiently label cultured muscle stem cells and allow for subsequent in vivo cell detection was determined. Magnetodendrimer-labeled cells exhibited normal growth rates in culture, and retained their capacity to undergo proliferation and form normal myotubes. Labeled stem cells possessed high in vivo proton relaxivities that enhanced MRI contrast properties and enabled us to noninvasively monitor the stem cells' incorporation into dystrophic muscle. Well defined regions of decreased signal intensity were observed in both T2- and T1-weighted image sequences. MRI was used to longitudinally follow stem cell dynamics in dystrophic muscle with in-plane resolutions on the order of a single muscle fiber (22 x 43 microm2). Regions of decreased signal intensity were well correlated with iron accumulation and other histochemical markers of stem cell incorporation. We concluded that this technique may be useful for continuous noninvasive readouts of stem cell transfer, replacing sequential muscle biopsies and tissue harvesting.

Animals↗
PubMed2004

Methionine sulfoxide reductase A (MsrA) deficiency affects the survival of Mycobacterium smegmatis within macrophages.

Methionine sulfoxide reductase A (MsrA) is an antioxidant repair enzyme which reduces oxidized methionine to methionine. Since oxidation of methionine in proteins impairs their function, an absence of MsrA leads to abnormalities in different organisms, including alterations in the adherence patterns and in vivo survival of certain pathogenic bacteria. To understand the role of MsrA in intracellular survival of bacteria, we disrupted the gene encoding MsrA in Mycobacterium smegmatis through homologous recombination. The msrA mutant strain of M. smegmatis exhibited significantly reduced intracellular survival in murine J774A.1 macrophages compared to the survival of its wild-type counterpart. Furthermore, immunofluorescence and immunoblotting of phagosomes containing M. smegmatis strains revealed that the phagosomes with the msrA mutant strain acquired both p67(phox) of phagocyte NADPH oxidase and inducible nitric oxide synthase much earlier than the phagosomes with the wild-type strain. In addition, the msrA mutant strain of M. smegmatis was observed to be more sensitive to hydroperoxides than the wild-type strain was in vitro. These results suggest that MsrA plays an important role in both extracellular and intracellular survival of M. smegmatis.

Animals↗
PubMed2003

Effects of the cowpea chlorotic mottle bromovirus beta-hexamer structure on virion assembly.

The X-ray crystal structure of Cowpea chlorotic mottle bromovirus (CCMV) revealed a unique tubular structure formed by the interaction of the N-termini from six coat protein subunits at each three-fold axis of the assembled virion. This structure, termed the beta-hexamer, consists of six short beta-strands. The beta-hexamer was postulated to play a critical role in the assembly and stability of the virion by stabilizing hexameric capsomers. Mutational analyses of the beta-hexamer structure, utilizing both in vitro and in vivo assembly assays, demonstrate that this structure is not required for virion formation devoid of nucleic acids in vitro or for RNA-containing virions in vivo. However, the beta-hexamer structure does contribute to virion stability in vitro and modulates disease expression in vivo. These results support a model for CCMV assembly through pentamer intermediates.

Base Sequence↗
PubMed2001

Ordered association of tobacco mosaic virus in the presence of divalent metal ions.

The formation of ordered aggregates of tobacco mosaic virus (TMV) in the presence of divalent metal ions has been studied in concentrated (1-25 mg/ml) solutions of the virus. The divalent metal cations Cd2+, Zn2+, Pb2+, Cu2+, and Ni2+ have been found to promote TMV precipitation from solution at a critical concentration Ccrit, which for a given metal depends on the pH and the ionic strength of the solution, but is largely independent of the virus concentration. The TMV precipitate behaves as a nematic liquid crystal and on drying at a glass surface produces highly ordered, optically birefringent films. However, precipitation is not observed with alkali-earth metals such as Ca2+ and Mg2+. The experimental data suggest that, apart from two 'internal' metal-binding sites in each TMV subunit, the virus contains metal-binding sites of a lower affinity which promote cross-linking of TMV rods via metal bridges. The latter seem to be responsible for the precipitation of TMV in the presence of divalent cations at neutral pH. We propose that the metal-induced cross-linking may be the predominant mechanism to account for the limited solubility of a variety of proteins in solution containing metal cations with valence 2 and higher.

Binding Sites↗
PubMed2001

Magnetodendrimers allow endosomal magnetic labeling and in vivo tracking of stem cells.

Magnetic resonance (MR) tracking of magnetically labeled stem and progenitor cells is an emerging technology, leading to an urgent need for magnetic probes that can make cells highly magnetic during their normal expansion in culture. We have developed magnetodendrimers as a versatile class of magnetic tags that can efficiently label mammalian cells, including human neural stem cells (NSCs) and mesenchymal stem cells (MSCs), through a nonspecific membrane adsorption process with subsequent intracellular (non-nuclear) localization in endosomes. The superparamagnetic iron oxide nanocomposites have been optimized to exhibit superior magnetic properties and to induce sufficient MR cell contrast at incubated doses as low as 1 microg iron/ml culture medium. When containing between 9 and 14 pg iron/cell, labeled cells exhibit an ex vivo nuclear magnetic resonance (NMR) relaxation rate (1/T2) as high as 24-39 s-1/mM iron. Labeled cells are unaffected in their viability and proliferating capacity, and labeled human NSCs differentiate normally into neurons. Furthermore, we show here that NSC-derived (and LacZ-transfected), magnetically labeled oligodendroglial progenitors can be readily detected in vivo at least as long as six weeks after transplantation, with an excellent correlation between the obtained MR contrast and staining for beta-galactosidase expression. The availability of magnetodendrimers opens up the possibility of MR tracking of a wide variety of (stem) cell transplants.

3T3 Cells↗
PubMed2000

Dynamic relaxometry: application to iron uptake by ferritin.

We introduce dynamic relaxometry as a novel technique for studying biochemical reactions, such as those leading to mineral formation (biomineralization). This technique was applied to follow the time course of iron oxidation and hydrolysis by the protein ferritin. Horse spleen apoferritin was loaded with single additions of 4, 10, 20, 40, and 100 ferrous ions per protein, and with multiple additions of 4, 10, 20, and 100 ferrous ions. The NMR T2 relaxation time was then measured sequentially and continuously for up to 24 h. At low loading factors of 4-10 Fe atoms/molecule, the iron is rapidly bound and oxidized by the protein on a time scale of approximately 15 s to several minutes. At intermediate loading factors (10-40), rapid initial oxidation was observed, followed by the formation of antiferromagnetic clusters. This process occurred at a much slower rate and continued for up to several hours, but was inhibited at lower pH values. At higher loading factors (40-1000), iron oxidation may occur directly on the core, and this process may continue for up to 24 h following the initial loading. Dynamic relaxometry appears to be a potentially powerful technique that may well have applications beyond the study of iron uptake by the ferritin protein.

Animals↗
PubMed2000

Effectiveness of lateral slide exercise in an anterior cruciate ligament reconstruction rehabilitation home exercise program.

STUDY DESIGN: Two-group repeated measures design using a sample of convenience of subjects with anterior cruciate ligament (ACL) reconstructive surgery. OBJECTIVES: To determine the effect of incorporating one specific weight-bearing exercise (lateral slide exercise using a slide board) into an ACL reconstruction home exercise program. BACKGROUND: Reduced clinic visits have increased the importance of home exercise programs in knee ligament reconstruction rehabilitation. Few studies have been conducted to test the efficacy of specific exercises as part of a home-based treatment program on subjects who have undergone ACL reconstruction. METHODS AND MEASURES: Fourteen subjects who underwent patella tendon autograft reconstruction on one of their ACLs were studied. Testing consisted of the following 4 measurements: peak isometric knee extension torque, peak isometric knee flexion torque, maximum lateral step height, and lateral step-up repetitions to fatigue. Subjects were pretested at 8 weeks after surgery and were randomly placed into either a control or experimental group. The postsurgical rehabilitation was similar for both groups, except the experimental group incorporated lateral slide exercise into their home exercise program. All subjects were re-evaluated 14 weeks after surgery. RESULTS: A 2-way repeated measure ANOVA (group by test session), and posthoc testing revealed significant improvements in the slide group for quadricep strength (101.9 +/- 31.3 N m to 140.5 +/- 31.3 N m of torque), while the control group showed no significant increase (125.1 +/- 61.7 N m to 125.8 +/- 45.1 N m of torque). Lateral step height also improved in the slide group (from 22.9 +/- 5.3 cm to 28.7 +/- 5.6 cm), while the control group showed no increase (20.0 +/- 4.5 cm to 20.7 +/- 3.4 cm). Both groups increased in lateral step-up repetitions to fatigue. CONCLUSION: Including lateral slide exercise in a home exercise program after ACL reconstruction appears to improve knee extension strength.

Adult↗
PubMed1999

Synthesis and characterization of hydrophobic ferritin proteins

Alkylated derivatives of the iron storage protein, ferritin, have been prepared by carbodiimide-activated coupling of long chain (C9, C12, C14) primary amines to surface carboxylic acid residues. In the case of a nonyl-derivatized protein, alkylation results in covalent modification of approximately 400 of the 520 amino acid carboxyl groups in the protein molecule. The hydrophobic proteins have a net positive charge in water and can be transferred from THF/water mixtures into dichloromethane, ethyl acetate and toluene by addition of small amounts of NaCl. Transmission electron microscopy, analytical ultracentrifugation, and polyacrylamide gel electrophoresis indicate that the hydrophobic proteins dissolve in the organic solvents as structurally intact, non-aggregated macromolecules which can be subsequently back-extracted into water.

Journal Article↗
PubMed1998

Calculated electrostatic gradients in recombinant human H-chain ferritin.

Calculations to determine the electrostatic potential of the iron storage protein ferritin, using the human H-chain homopolymer (HuHF), reveal novel aspects of the protein. Some of the charge density correlates well with regions previously identified as active sites in the protein. The three-fold channels, the putative ferroxidase sites, and the nucleation sites all show expectedly negative values of the electrostatic potential. However, the outer entrance to the three-fold channels are surrounded by regions of positive potential, creating an electrostatic field directed toward the interior cavity. This electrostatic gradient provides a guidance mechanism for cations entering the protein cavity, indicating the three-fold channel as the major entrance to the protein. Pathways from the three-fold channels, indicated by electrostatic gradients on the inner surface, lead to the ferroxidase center, the nucleation center and to the interior entrance to the four-fold channel. Six glutamic acid residues at the nucleation site give rise to a region of very negative potential, surrounding a small positively charged center due to the presence of two conserved arginine residues, R63, in close proximity (4.9 A), suggesting that electrostatic fields could also play a role in the nucleation process. A large gradient in the electrostatic potential at the 4-fold channel gives rise to a field directed outward from the internal cavity, indicating the possibility that this channel functions to expel cations from inside the protein. The 4-fold channel could therefore provide an exit pathway for protons during mineralization, or iron leaving the protein cavity during de-mineralization.

Ceruloplasmin↗
PubMed1998

Measurement of serum prostate-specific membrane antigen, a new prognostic marker for prostate cancer.

OBJECTIVES: To describe current results with Western blot assay for prostate specific membrane antigen (PSMA) using 7E11.C5 antibody and the development of an additional antibody measurement for PSMA by a new sandwich immunoassay. METHODS: A population of patients from a screening group, from a difficult diagnostic group, from a pre- and postoperative radical prostatectomy group, and from a group with metastatic disease followed for a serial period, provided the serum values for a prospective assessment of PSMA by Western blot assay. A new monoclonal antibody was sought, reacting to the C-terminal region of PSMA in order to develop a sandwich radioimmunoassay. RESULTS: PSMA values in screened patients correlate with the more advanced stage of the cancers determined. In postprostatectomy patients, the PSMA value corresponds more with preoperative values and with the values of those with a poor clinical course. In difficult diagnostic cases, the PSMA value is increased, specifically in hormone-refractory cases and particularly in those cases judged by other criteria, such as the National Prostatic Cancer Project, to be in clinical progression compared with those judged to be in clinical remission. The level of PSMA value appears to be independent of homogeneous tumor volume and to be more related to that of prior hormone treatment, or to where prostate cancer cells can be documented to be outside the prostate. A new monoclonal antibody, 3F5.4G6, reacts with the extracellular domain of PSMA near the C-terminal region. This is in contrast to the previously measured antibody 7E11.C5, which reacts with an N-terminal epitope. 3F5.4G6 recognizes the same PSMA protein as does 7E11.C5. The epitopes are essentially at opposite ends of the molecule. The 3F5.4G6 antibody reacts with the LNCaP line but not with DU145, or PC3. These two antibodies to PSMA are well suited for use in a new sandwich immunoassay. CONCLUSIONS: PSMA provides a prostatic cancer serum test by using Western blot, which suggests a clinical prognostic value not seen with other markers. New antibodies, such as 3F5.4G6, reacting with the extracellular domain of PSMA combined with 7E11.C5, appear to offer an opportunity for a new sandwich immunoassay.

Antibodies, Monoclonal↗
PubMed1997

"Scalded mouth syndrome" caused by angiotensin converting enzyme inhibitors: two case reports.

Two cases of "scalded mouth syndrome" are presented and discussed. This condition is a rare side effect of angiotensin converting enzyme inhibitors. Angiotensin converting enzyme inhibitors are known to cause several other oral and extra-oral side effects. In scalded mouth syndrome, the patient's complaints concern a burning pain of the oral soft tissues. There are no clinical findings.

Aged↗
PubMed1997

Salmonella infection acquired from reptilian pets.

Two children presented with signs and symptoms of gastroenteritis. Salmonella chameleon was isolated from the stool of one child and also from an iguana kept in the home as a pet. Salmonella arizonae was isolated from the stool of the other child and also from four snakes sharing the same household. Exotic reptiles are unsuitable pets to share the home environment with infants.

Animals↗
PubMed1996

Evaluation and comparison of two new prostate carcinoma markers. Free-prostate specific antigen and prostate specific membrane antigen.

BACKGROUND: Two new prostate cancer markers, free-prostate specific antigen (f-PSA) and prostate specific membrane antigen (PSMA) were recently introduced. This report summarizes a prospective two-year multicenter test of their diagnostic or prognostic capabilities. Total PSA was also measured. METHODS: There were four clinical groups studied: (1) 226 individuals from a screening project undergoing ultrasound and biopsy evaluation had markers obtained: (2) 68 patients suspected of having prostate cancer and undergoing 2 or more biopsies had the markers obtained on multiple occasions: (3) 100 patients undergoing radical prostatectomy had markers obtained pre- and post-operatively: and (4) 31 patients with metastatic prostate cancer each had multiple samples for marker assay obtained over a 2-year period. In all, 465 patients had one or more samples obtained and studied. RESULTS: Free-PSA affords little additional diagnostic advantage compared with total PSA in the screening population. The receiver operating characteristic curves for diagnostic accuracy were ranked: (1) PSA density; (2) total PSA; (3) f-PSA; and (4) PSMA, PSMA showed the best correlation with stage of the primary tumor in the screened group. In the multiple negative biopsy group, f-PSA varied from 12 to 21%. PSMA values were evaluated in all histologic categories. PSA density was > or = 0.15 in all categories. In the prostatectomy cases PSA values postoperatively were quite low in Stage II; f-PSA was of no value. Later, f-PSA was increased in association with elevated total PSA values. Mean PSMA values were above normal in all postoperative time periods except in Stage III patients at 6 months to 1 year postoperatively. PSA densities were all > or = 0.15. In patients with metastatic carcinoma, elevated PSMA values correlated best with a poor prognosis (clinical progression), as has been described. CONCLUSIONS: These data suggest that f-PSA values do not provide additional diagnostic benefit compared with total PSA in screening populations, in the presence of suspected cancer, postprostatectomy, or in metastatic disease. PSMA is of prognostic significance, especially in the presence of metastatic disease, and correlates well with the stage of disease in cancers detected in a screened population.

Antigens, Neoplasm↗