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PubMed2006

Mycoplasma genitalium P140 and P110 cytadhesins are reciprocally stabilized and required for cell adhesion and terminal-organelle development.

Mycoplasma genitalium is a human pathogen that mediates cell adhesion by a complex structure known as the attachment organelle. This structure is composed of cytadhesins and cytadherence-associated proteins, but few data are available about the specific role of these proteins in M. genitalium cytadherence. We have deleted by homologous recombination the mg191 and mg192 genes from the MgPa operon encoding the P140 and P110 cytadhesins. Molecular characterization of these mutants has revealed a reciprocal posttranslational stabilization between the two proteins. Loss of either P140 or P110 yields a hemadsorption-negative phenotype and correlates with decreased or increased levels of cytoskeleton-related proteins MG386 and DnaK, respectively. Scanning electron microscopy analysis reveals the absolute requirement of P140 and P110 for the proper development of the attachment organelle. The phenotype described for these mutants resembles that of the spontaneous class I and class II cytadherence-negative mutants [G. R. Mernaugh, S. F. Dallo, S. C. Holt, and J. B. Baseman, Clin. Infect. Dis. 17(Suppl. 1):S69-S78, 1993], whose genetic basis remained undetermined until now. Complementation assays and sequencing analysis demonstrate that class I and class II mutants are the consequence of large deletions affecting the mg192 and mg191-mg192 genes, respectively. These deletions originated from single-recombination events involving sequences of the MgPa operon and the MgPa island located immediately downstream. We also demonstrate the translocation of MgPa sequences to a particular MgPa island by double-crossover events. Based on these observations, we propose that in addition to being a source of antigenic variation, MgPa islands could be also involved in a general phase variation mechanism switching on and off, in a reversible or irreversible way, the adhesion properties of M. genitalium.

Adhesins, Bacterial↗
PubMed1987

Initial studies of a phytoestrogen-deoxyribonucleic acid interaction.

Molecular modeling studies show that estrogens such as estradiol complement the topography of spaces between base pairs in unwound DNA and simultaneously hydrogen bond phosphate moieties on opposite strands. We demonstrate here that the phytoestrogen coumestrol has this capability, in addition to its documented properties of UV absorbance at lambda greater than 300 nm and fluorescence. The latter properties enable spectroscopic examination of interactions with DNA by methods not possible with estrogenic steroids. On exposure to calf thymus DNA, the UV spectrum of coumestrol displays a bathochromic shift and simultaneous hypochromic effect with an isosbestic point at 370 nm, suggesting a shift between coexisting free and bound states. Similar results are observed with the intercalating agents adriamycin, ethidium bromide, and acridine. The fluorescence spectrum of coumestrol is quenched on exposure to DNA as are those of adriamycin and acridine. Coumestrol differs from the intercalators in that denatured DNA does not affect its UV spectrum or alter its relative fluorescence yield. Unlike classical intercalators, coumestrol has no influence on the thermal stability of calf thymus DNA. Preliminary electrophoretic analysis of DNA plasmid conformers indicates that coumestrol is incapable of significantly altering DNA superhelical density, in contrast to ethidium bromide. These initial physicochemical data provide evidence for the DNA base-estrogen electronic and/or hydrophobic interactions suggested by modeling studies, yet tend to rule out classical intercalation as an explanation for these phenomena.

Animals↗
PubMed2003

A quantitative analysis of intron effects on mammalian gene expression.

In higher eukaryotes, intron-containing and intronless versions of otherwise identical genes can exhibit dramatically different expression profiles. Introns and the act of their removal by the spliceosome can affect gene expression at many different levels, including transcription, polyadenylation, mRNA export, translational efficiency, and the rate of mRNA decay. However, the extent to which each of these steps contributes to the overall effect of any one intron on gene expression has not been rigorously tested. Here we report construction and initial characterization of a luciferase-based reporter system for monitoring the effects of individual introns and their position within the gene on protein expression in mammalian cells. Quantitative analysis of constructs containing human TPI intron 6 at two different positions within the Renilla luciferase open reading frame revealed that this intron acts primarily to enhance mRNA accumulation. Spliced mRNAs also exhibited higher translational yields than did intronless transcripts. However, nucleocytoplasmic mRNA distribution and mRNA stability were largely unaffected. These findings were extended to two other introns in a TCR-beta minigene.

Animals↗
PubMed2005

Nanostructured DNA templates.

We have developed methods for nanostructure fabrication relying on the size and shape of a polynucleotide to dictate the overall structure of an assemblage of individual semiconductor nanoparticles. Use of the circular plasmids pUCLeu4 and phi chi 174 when anchored to a suitably derivatized substrate yields arrays of semiconductor nanoparticles matching the shapes of the biopolymer stabilizer. The viability of the methodology was confirmed using high-resolution transmission electron microscopy and selected-area electron diffraction.

Nanostructures↗
PubMed2003

Growth hormone-releasing hormone and growth hormone secretagogues in normal aging.

Growth hormone (GH) secretion declines with aging, and parallels between normal aging and the signs and symptoms of adult GH deficiency have led to interest in the potential utility of replacing or stimulating GH to promote physical and psychological function and to prolong the capacity for independent living in older adults. The aging pituitary remains responsive to GH-releasing hormone (GHRH) and to ghrelin-mimetic GH secretagogues (GHS), and these agents have both theoretical and practical potential advantages as alternatives to the use of GH itself in this setting. Studies of the long duration and large scale needed to test the efficacy of GHRH or GHS on clinically important endpoints cannot be designed or conducted without first obtaining promising results in studies of smaller size focused on manageable intermediate endpoints, and all studies published to date have been of this latter type. GHRH and GHS both stimulate GH secretion, and, when given repeatedly, elevate IGF-I levels to within younger adult normal ranges. When GHRH treatment is continued for several months, these hormonal changes yield an increase in lean body (muscle) mass. GHRH, like GH, reduces body fat, but similar effects have not yet been shown with GHS. GHRH treatment has not yielded consistent improvements in physical function, although it may have a stabilizing effect. Chronic treatment with a short-acting GHRH did not improve sleep, possibly due to lack of sustained activity throughout the night. Compared to placebo, GHRH treatment improved certain tests of cognitive performance. These results, while encouraging, do not yet support the routine use of GHRH or GHS in normal aging.

Aging↗
PubMed2002

Synthesis, spectra and photophysics of some free base tetrafluoroalkyl and tetrafluoroaryl porphyrins with potential applications in imaging.

The synthesis, characterization and photophysical properties of two perfluoroalkyl (5,10,15,20-tetrakis-[trifluoromethyl]- and [heptafluoropropyl]-porphyrin) and two perfluoroaryl (5,10,15,20-tetrakis-[2,6-difluorophenyl]- and [pentafluorophenyl]-porphyrin) are described, with reference to their potential in both photodynamic therapy (PDT) and in vivo imaging by fluorescence and 19F nuclear magnetic resonance spectroscopy. Absorption and fluorescence spectra, fluorescence lifetimes and triplet-singlet difference spectra are reported. Triplet yields have been obtained by flash photolysis and pulse radiolysis, whereas yields of sensitized singlet oxygen formation have been determined by time-resolved phosphorimetry. All four compounds show high yields of triplet formation and singlet oxygen sensitization. The spectral properties, stability and attractive solubility characteristics of the perfluoroalkyl derivatives make them particularly suitable candidates for future study for applications in PDT.

Magnetic Resonance Spectroscopy↗
PubMed1995

Evaluation of varied surgical approaches used in the management of 170 far-lateral lumbar disc herniations: indications and results.

This study was undertaken to determine and compare indications and relative benefits of various surgical approaches in 170 patients (average age 55 years) with far-lateral herniated lumbar discs, identified by magnetic resonance (MR) imaging and computerized tomography (CT) and operated on between 1984 and 1994. Essentially three surgical procedures were performed: complete facetectomy in 73 patients, laminotomy with medial facetectomy in 39 patients, and intertransverse discectomy (also known as ITT) in 58 patients. Follow-up periods averaged 5 years (range 0.5-10 years). Outcomes were scored as excellent (no deficit), good (mild radiculopathy), fair (moderate radiculopathy), and poor (unchanged or worse). Overall, excellent and good results were achieved in 73 and 51 patients, respectively, and fair and poor results in 26 and 20, respectively. There was little difference among the results encountered for the three major surgical groups: 79% of the intertransverse (ITT) group had good-to-excellent outcomes, as compared with 70% of the facetectomy group, and 68% of the group who underwent at minimum laminotomy, and additional hemilaminectomy or laminectomy with medial facetectomy. Results were the same for the 121 patients followed for more than 2 years and for the 49 patients studied for under 2 years. In the management of far-lateral discs, total facetectomy provides the best exposure, but increases the risk of instability. Laminotomy and medial facetectomy uncover the lateral and subarticular recess and preserve stability, but visualization of the far-lateral compartment is often inadequate. The intertransverse approach offers extensive far-lateral but not medial intraforaminal exposure, while also preserving stability. Full facetectomy, laminotomy with medial facetectomy, and the intertransverse approaches yielded nearly comparable outcomes in far-lateral disc surgery. Only the full facetectomy exposes the entire course of the nerve root both medially and laterally, whereas the intertransverse procedure provides direct exposure of the fat-lateral compartment alone. It is important to select the correct approach or combination of approaches to address attendant complicating factors such as spinal stenosis, spondyloarthrosis, and degenerative spondylolisthesis identified on CT and MR studies.

Adult↗
PubMed2001

Transition Metal Catalysis Using Functionalized Dendrimers.

Dendrimers are well-defined hyperbranched macromolecules with characteristic globular structures for the larger systems. These novel polymers have inspired many chemists to develop new materials and several applications have been explored, catalysis being one of them. The recent impressive strides in synthetic procedures increased the accessibility of functionalized dendrimers, resulting in a rapid development of dendrimer chemistry. The position of the catalytic site(s) as well as the spatial separation of the catalysts appears to be of crucial importance. Dendrimers that are functionalized with transition metals in the core potentially can mimic the properties of enzymes, their efficient natural counterparts, whereas the surface-functionalized systems have been proposed to fill the gap between homogeneous and heterogeneous catalysis. This might yield superior catalysts with novel properties, that is, special reactivity or stability. Both the core and periphery strategies lead to catalysts that are sufficiently larger than most substrates and products, thus separation by modern membrane separation techniques can be applied. These novel homogeneous catalysts can be used in continuous membrane reactors, which will have major advantages particularly for reactions that benefit from low substrate concentrations or suffer from side reactions of the product. Here we review the recent progress and breakthroughs made with these promising novel transition metal functionalized dendrimers that are used as catalysts, and we will discuss the architectural concepts that have been applied.

Journal Article↗
PubMed2002

An improved assay for the determination of C-reactive protein.

C-reactive protein as the most important acute phase reactant in clinical use provides information about acute as well as chronic inflammatory processes. This application requires an improvement of traditional assays that have been available to the clinical laboratory regarding lipemic interference, precision especially at the lower end, assay and calibration stability as well. In the present study the improved Olympus turbidimetric assay for the determination of C-reactive protein (CRP) was evaluated on the Olympus AU640 analytical system. The assay has a lower detection limit of 1.57 mg/l CRP and is linear from 5 mg/l to 200 mg/l. Prozone hook effect did not occur until 300 mg/l with increased prozone sample detection to 3500 mg/l. Imprecison CV values for within-run of less than 2.25% and between-day of lower than 3.1% were found. On-board stability was extended to 60 days, and calibration stability to 28 days. Method comparison to another automated CRP assay yielded a correlation coefficient of r=0.999. Endogenous substances did not interfere with the test results. There was satisfactory recovery of target values according to CRM 470 standardization. In comparison to the previous assay the improved assay offers more accuracy, precision, and linearity and is free from any known interference, which meets user needs, for rapid and convenient determination of CRP on automated analyzers.

C-Reactive Protein↗
PubMed2004

MetMAP: an integrated Matlab package for analysis and optimization of metabolic systems.

In previous works we have presented and applied a method to predict the parameter profile that optimizes biochemical systems regarding either a single or a set of metabolic responses within physiological constraints [Vera et al., 2003a]. This optimization technique requires a previous model definition and a translation to S-system form and the use of widely available linear programming packages. However, in dealing with these issues the interested researcher has to confront additional difficulties because of a lack of connectivity among available software packages or routines specifically designed to perform different tasks. In addition to this difficulty is the unavailability of any automated package which is capable of performing such optimizations and the previous required analysis. This situation prompted us to develop an integrated software package able to deal with these tasks in a single program environment. In this paper we present a software package for the model definition, analysis and optimization of a biochemical system. It starts with a given model definition that is directly translated to its equivalent S-system form. Once the model quality assessment is performed (stability and sensitivity analysis) the program determines the parameter profile that yields the optimized response compatible with a predefined set of constraints. Moreover the package finds the set of solutions obtained when more than one system's responses are to be optimized (multiobjective optimization).

Algorithms↗
PubMedDate not supplied

[Modified EMJH medium for cultivation of Leptospira interrogans serogroup ballum].

Strains within the Ballum serogroup of spirochete Leptospira show fastidious growth with more exigent nutritional requirements than those of other Leptospira pathogenic strains. The influence of 37 nutritional compounds on the growth of Leptospira interrogans serogroup Ballum was investigated employing the synthetic EMJH medium as the base for the study. Microbial growth was estimated spectrophotometrically and direct counts were performed with a Petroff-Hausser counting chamber. Virulence stability was evaluated by calculating the mean lethal dose in hamsters. Antigenicity stability was evaluated by Western blotting using a specific antiserum. Cell yields commonly obtained in EMJH were triplicated without virulence or antigenicity depletions after culturing in a modified EMJH medium with an increased concentration of Tween 80, and the incorporation of sodium acetate and beef extract. Neither the increased concentration of at least 6 components of EMJH nor the incorporation of a variety of new nutrients stimulated cell yields or the growth rate of the microorganism. The results allow us to make use of an enriched culture medium that promotes high cell yields of this fastidious serogroup most prevalent in humans in Cuba.

Culture Media↗
PubMed1991

[Chemical reactions in double helical nucleic acids. XIII. Directed introduction of acylphosphate internucleotide bonds into the DNA-duplex structure].

DNA duplex, containing an acylphosphate internucleotide bond in a predetermined position of the sugar-phosphate backbone, was synthesized. The synthesis was carried out by condensing on the complementary matrix two heptanucleotides, one of which possessed at the 3'-end a glycine residue, connected with the oligonucleotide by the phosphoramide bond, whereas the 5'-end phosphate group of the other was activated with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDAC). The yield of the oligonucleotide with an acylphosphate bond was 24%. The stability and chemical properties of the synthesized compound were studied in comparison with analogous oligonucleotide containing a substituted pyrophosphate internucleotide bond. The former was shown to be an effective acylating agent in the aqueous medium in contrast to the latter which is a phosphorylating agent.

Acylation↗
PubMed1990

Use of plasma with high levels of ionised calcium in the production of model scale coagulation factor concentrates.

We have attempted to exploit the Ca2(+)-dependent stability of factor VIII in producing factor VIII concentrates of higher yield. Plasma levels of ionised calcium were increased in two ways: (a) whole blood collection into half-strength citrate CPD anticoagulant, leading to free Ca2+ levels of ca 120 microM and (b) apheresis collection of plasma which was then recalcified to free Ca2+ levels of ca 300 microM under heparin cover. Coagulation factor concentrates were prepared using model versions of our industrial scale manufacturing methods. Factor VIII yield was increased through low citrate collection. This did not compromise factor IX yield or thrombogenic potential. Use of recalcified heparinised plasma did not lead to any improvement in factor VIII yield and resulted in a marked drop in factor IX recovery, possibly from interference by heparin of factor IX binding in ion-exchange chromatography. The benefits accruable through the use of half-strength citrate CPD anticoagulant support the continued evaluation of this preservative in large scale blood collection and fractionation. The deleterious effects of heparin in charge-mediated plasma fractionations may pose serious difficulties in harvesting vitamin K dependent factors.

Anticoagulants↗
PubMed1990

[Development of passive hemagglutination assay of measles and utilization in serological diagnosis].

The passive hemagglutination assay (PHA) was developed. Preparation of antigen and its stability, sensitivity and specificity was studied. The result showed that the yield of ultrasonic treated antigen is high and its titer has no change in 4 degrees C storage at least 1 month. The sensitized sheep erythrocyte with this antigen have a high specificity and sensitivity. The lysophilized blood cells could stored for a long time in 4 degrees C. A comparison of PHA with hemagglutination inhibition test was carried out by examination of 354 samples of sera from healthy children. The GMT of titer of antibodies of measles were 26.20 and 13.92 respectively. The result showed that the two methods were highly related.

Antigens, Viral↗
PubMed1989

[Benzidine and its derivatives in the immunohistochemical analysis of peroxidase].

Authors have studied the application of benzidine and its derivatives for the demonstration of the marker enzyme horseradish peroxidase. Benzidine compounds are known to be in a blueish dimeric form under oxidative circumstances, while after polymerization they yield a dark brown end product. If the dimeric state is stabilized, the 3,3'-dimethoxy benzidine (o-dianisidin) appears to be the most suitable to demonstrate peroxidase. The o-dianisidin reaction has the advantage of high sensitivity and that the end product shows a birefringence in polarized light. Upon mounting with organic media the end product is stable.

Benzidines↗
PubMed1984

Monitoring exposure to 4-aminobiphenyl via blood protein adducts.

The feasibility of monitoring exposure to 4-aminobiphenyl (4-ABP) was determined by measuring the formation of covalent blood protein adducts in rats following administration of the amine. A single major adduct was obtained from serum albumin, which was formed from 4-acetylaminobiphenyl and the single tryptophan residue of albumin. The adduct was isolated as part of a tetrapeptide, following Pronase digestion and reverse-phase high-performance liquid chromatography purification, and was identified by amino acid analysis, 1H-nuclear magnetic resonance and mass spectrometry. This adduct was cleared in vivo and showed a half-life of 4.7 days, essentially identical to that of albumin. Similarly, 4-ABP yielded a single major adduct with haemoglobin. This adduct showed a stability in vivo that was comparable to that of haemoglobin, and was shown to accumulate, with chronic dosing, to a level 30 times higher than that resulting from a single dose. Treatment of adducted haemoglobin, with 0.1 N hydrochloric acid in acetone, caused hydrolysis and release of 4-ABP. The formation of the haemoglobin adduct involves 4-nitrosobiphenyl as an intermediate product and, by implication, 4-hydroxyaminobiphenyl. Thus, the haemoglobin adduct is a measure of the fraction of 4-ABP that is N-hydroxylated directly, whereas the albumin adduct reflects that portion which is acetylated prior to N-hydroxylation. Approximately 0.02% of a dose of 4-ABP was bound to the tryptophan in albumin (dose of 2-80 mg/kg) and 5% was bound to haemoglobin as an acid-labile adduct (dose of 0.5-5000 micrograms/kg).

Acetylation↗
PubMed1983

The role of arginyl residues in estrogen receptor activation and transformation.

Receptor-estradiol complexes (RE2) formed at 0 degree C in hypotonic buffers bind poorly to nuclei (nonactivated state); their sedimentation coefficient in low or high salt sucrose density gradients (SDG) is 8 S or 4 S, respectively (untransformed state); estradiol dissociates from untransformed RE2 at a high rate (k-1 = 0.44 min-1). Brief heating (28 degrees C, 30 min) induces activation (increased binding of RE2 to nuclei and polyanions), transformation (formation of receptor dimers which sediment at 6 S in 0.4 M KCl/borate SDG) and RE2 transition into a state from which E2 dissociates at a lower rate (k-2 = 8 X 10(-3) min-1). We have examined the role of arginyl residues in the above changes in receptor properties. It is well established (Patthy, L., and Smith, E. L. (1975) J. Biol. Chem. 250, 557-564; 565-569) that 1,2-cyclohexanedione (1,2-CHD) is a highly specific arginine-modifying agent; in borate buffer at 28 degrees C, but not at 0 degrees C, peptide arginyls are covalently modified. RE2 complexes heated in the presence of 1,2-CHD (50 mM) bind poorly to nuclei; 1,4-cyclohexamedione and 1,2-cyclohexanediol had no effect. This reagent also prevents the temperature-induced transition of RE2 into a state with slow E2 dissociation rates although it does not interfere with heat transformation (formation of 6 S dimer). Modification of heat-activated and transformed RE2 by 1,2-CHD causes a loss in receptor binding to nuclei and alters RE2 from a state with slow into a state with fast E2 dissociation rates, although the receptor remains unaltered in the transformed 6 S state. At 0 degree C, i.e. in the absence of covalent arginyl modification, 1,2-CHD promotes dissociation of the 8 S aggregate into 4.6 S subunits which bind to nuclei to the same extent as heat-transformed control RE2. Heating of the molybdate-stabilized 8 S receptor in the presence of 1,2-CHD yields a nonactivated 8 S receptor (4.6 S on high salt SDG); removal of molybdate and unreacted 1,2-CHD by gel filtration at 0 degree C followed by exposure to high ionic strength causes 8 S to 4 S dissociation; these 4 S subunits, however, do not bind to nuclei, suggesting that their nucleotropic domain was accessible to 1,2-CHD modification while the receptor was in the aggregated 8 S state. It is proposed that the nuclear binding site of the estrogen receptor contains arginyl residues. Furthermore, a distinct set of arginyl residues appears to be related to the estrogen-binding domain; its integrity is required for the heat-induced formation and maintenance of the RE2 state with slow E2 dissociation.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1983

Enhanced tumorigenicity of cloned UV-regressor tumor lines following selected in vivo and in vitro manipulations.

Passage of cloned ultraviolet (UV) radiation-induced fibrosarcomas with regressor phenotype through 500-rad-irradiated syngeneic mice resulted in their conversion to transplantable progressor tumors. A similar conversion in tumorigenic phenotype (regressor leads to progressor) was found to be inducible in vitro by coculturing a cloned regressor tumor with normal splenocytes, but not with splenocytes from tumor-immune or UV-irradiated animals. Recloning of regressor and converted progressor tumor lines yielded regressor and progressor phenotype subclones, respectively, suggesting a degree of stability in their growth phenotype. Although all of the cloned progressor tumors tested were found to be cross-reactive with related regressor tumor lines, suggesting that related clones share a similar tumor-specific transplantation antigen, the progressor clones appeared to be less immunogenic than the regressor clones. Potential mechanisms that influence this conversion in tumorigenic phenotype are discussed.

Animals↗