Monolayers of silver nanoparticles decrease photobleaching: application to muscle myofibrils.

Studying single molecules in a cell has the essential advantage that kinetic information is not averaged out. However, since fluorescence is faint, such studies require that the sample be illuminated with the intense light beam. This causes photodamage of labeled proteins and rapid photobleaching of the fluorophores. Here, we show that a substantial reduction of these types of photodamage can be achieved by imaging samples on coverslips coated with monolayers of silver nanoparticles. The mechanism responsible for this effect is the interaction of localized surface plasmon polaritons excited in the metallic nanoparticles with the transition dipoles of fluorophores of a sample. This leads to a significant enhancement of fluorescence and a decrease of fluorescence lifetime of a fluorophore. Enhancement of fluorescence leads to the reduction of photodamage, because the sample can be illuminated with a dim light, and decrease of fluorescence lifetime leads to reduction of photobleaching because the fluorophore spends less time in the excited state, where it is susceptible to oxygen attack. Fluorescence enhancement and reduction of photobleaching on rough metallic surfaces are usually accompanied by a loss of optical resolution due to refraction of light by particles. In the case of monolayers of silver nanoparticles, however, the surface is smooth and glossy. The fluorescence enhancement and the reduction of photobleaching are achieved without sacrificing the optical resolution of a microscope. Skeletal muscle myofibrils were used as an example, because they contain submicron structures conveniently used to define optical resolution. Small nanoparticles (diameter approximately 60 nm) did not cause loss of optical resolution, and they enhanced fluorescence approximately 500-fold and caused the appearance of a major picosecond component of lifetime decay. As a result, the sample photobleached approximately 20-fold more slowly than the sample on glass coverslips.

Cross-bridge duty cycle in isometric contraction of skeletal myofibrils.

During interaction of actin with myosin, cross-bridges impart mechanical impulses to thin filaments resulting in rotations of actin monomers. Impulses are delivered on the average every tc seconds. A cross-bridge spends a fraction of this time (ts) strongly attached to actin, during which it generates force. The "duty cycle" (DC), defined as the fraction of the total cross-bridge cycle that myosin spends attached to actin in a force generating state (ts/ tc), is small for cross-bridges acting against zero load, like freely shortening muscle, and increases as the load rises. Here we report, for the first time, an attempt to measure DC of a single cross-bridge in muscle. A single actin molecule in a half-sarcomere was labeled with fluorescent phalloidin. Its orientation was measured by monitoring intensity of the polarized TIRF images. Actin changed orientation when a cross-bridge bound to it. During isometric contraction, but not during rigor, actin orientation oscillated between two values, corresponding to the actin-bound and actin-free state of the cross-bridge. The average ts and tc were 3.4 and 6 s, respectively. These results suggest that, in isometrically working muscle, cross-bridges spend about half of the cycle time attached to actin. The fact that 1/ tc was much smaller than the ATPase rate suggests that the bulk of the energy of ATP hydrolysis is used for purposes other than performance of mechanical work.

Decreasing photobleaching by silver island films: application to muscle.

Recently it has become possible to study interactions between proteins at the level of single molecules. This requires collecting data from an extremely small volume, small enough to contain one molecule-typically of the order of attoliters (10(-18) L). Collection of data from such a small volume with sufficiently high signal-to-noise ratio requires that the rate of photon detection per molecule be high. This calls for a large illuminating light flux, which in turn leads to rapid photobleaching of the fluorophores that are labeling the proteins. To decrease photobleaching, we measured fluorescence from a sample placed on coverslips coated with silver island films (SIF). SIF reduce photobleaching because they enhance fluorescence brightness and significantly decrease fluorescence lifetime. Increase in the brightness effectively decreases photobleaching because illumination can be attenuated to obtain the same fluorescence intensity. Decrease of lifetime decreases photobleaching because short lifetime minimizes the probability of oxygen attack while the fluorophore is in the excited state. The decrease of photobleaching was demonstrated in skeletal muscle. Myofibrils were labeled lightly with rhodamine-phalloidin, placed on coverslips coated with SIF, illuminated by total internal reflection, and observed through a confocal aperture. We show that SIF causes the intensity of phalloidin fluorescence to increase 4-5 fold and its fluorescence lifetime to decrease on average 23-fold. As a consequence, the rate of photobleaching of four or five molecules of actin of a myofibril on Olympus coverslips coated with SIF decreased at least 30-fold in comparison with photobleaching on an uncoated coverslip. Significant decrease of photobleaching makes the measurement of signal from a single cross-bridge of contracting muscle feasible.

E22K mutation of RLC that causes familial hypertrophic cardiomyopathy in heterozygous mouse myocardium: effect on cross-bridge kinetics.

Familial hypertrophic cardiomyopathy is a disease characterized by left ventricular and/or septal hypertrophy and myofibrillar disarray. It is caused by mutations in sarcomeric proteins, including the ventricular isoform of myosin regulatory light chain (RLC). The E22K mutation is located in the RLC Ca(2+)-binding site. We have studied transgenic (Tg) mouse cardiac myofibrils during single-turnover contraction to examine the influence of E22K mutation on 1) dissociation time (tau(1)) of myosin heads from thin filaments, 2) rebinding time (tau(2)) of the cross bridges to actin, and 3) dissociation time (tau(3)) of ADP from the active site of myosin. tau(1) was determined from the increase in the rate of rotation of actin monomer to which a cross bridge was bound. tau(2) was determined from the rate of anisotropy change of the recombinant essential light chain of myosin labeled with rhodamine exchanged for native light chain (LC1) in the cardiac myofibrils. tau(3) was determined from anisotropy of muscle preloaded with a stoichiometric amount of fluorescent ADP. Cross bridges were induced to undergo a single detachment-attachment cycle by a precise delivery of stoichiometric ATP from a caged precursor. The times were measured in Tg-mutated (Tg-m) heart myofibrils overexpressing the E22K mutation of human cardiac RLC. Tg wild-type (Tg-wt) and non-Tg muscles acted as controls. tau(1) was statistically greater in Tg-m than in controls. tau(2) was shorter in Tg-m than in non-Tg, but the same as in Tg-wt. tau(3) was the same in Tg-m and controls. To determine whether the difference in tau(1) was due to intrinsic difference in myosin, we estimated binding of Tg-m and Tg-wt myosin to fluorescently labeled actin by measuring fluorescent lifetime and time-resolved anisotropy. No difference in binding was observed. These results suggest that the E22K mutation has no effect on mechanical properties of cross bridges. The slight increase in tau(1) was probably caused by myofibrillar disarray. The decrease in tau(2) of Tg hearts was probably caused by replacement of the mouse RLC for the human isoform in the Tg mice.

Rotations of a few cross-bridges in muscle by confocal total internal reflection microscopy.

In order to measure the cycling of a few ( approximately 6) myosin heads in contracting skeletal muscle, myofibrils were illuminated by Total Internal Reflection and observed through a confocal aperture. Myosin heads rotated at a rate approximately equal to the ATPase rate, suggesting that bulk ATPase of a whole muscle reflects the cycle frequency of individual heads.

Changes in orientation of actin during contraction of muscle.

It is well documented that muscle contraction results from cyclic rotations of actin-bound myosin cross-bridges. The role of actin is hypothesized to be limited to accelerating phosphate release from myosin and to serving as a rigid substrate for cross-bridge rotations. To test this hypothesis, we have measured actin rotations during contraction of a skeletal muscle. Actin filaments of rabbit psoas fiber were labeled with rhodamine-phalloidin. Muscle contraction was induced by a pulse of ATP photogenerated from caged precursor. ATP induced a single turnover of cross-bridges. The rotations were measured by anisotropy of fluorescence originating from a small volume defined by a narrow aperture of a confocal microscope. The anisotropy of phalloidin-actin changed rapidly at first and was followed by a slow relaxation to a steady-state value. The kinetics of orientation changes of actin and myosin were the same. Extracting myosin abolished anisotropy changes. To test whether the rotation of actin was imposed by cross-bridges or whether it reflected hydrolytic activity of actin itself, we labeled actin with fluorescent ADP. The time-course of anisotropy change of fluorescent nucleotide was similar to that of phalloidin-actin. These results suggest that orientation changes of actin are caused by dissociation and rebinding of myosin cross-bridges, and that during contraction, nucleotide does not dissociate from actin.

Simultaneous measurement of rotations of myosin, actin and ADP in a contracting skeletal muscle fiber.

The rotation of myosin heads and actin were measured simultaneously with an indicator of the enzymatic activity of myosin. To minimize complications due to averaging of signals from many molecules, the signal was measured in a small population residing in a femtoliter volume of a muscle fiber. The onset of rotation was synchronized by a sudden release of caged ATP. The orientation of cross-bridges was measured by anisotropy of recombinant fluorescent regulatory light chains exchanged with native regulatory light chains. The orientation of actin was measured by anisotropy of phalloidin added to actin filaments. The enzymatic activity of myosin was measured by dissociation of fluorescent ADP from the active site. The onset of all three events occurred at the same time. This suggests that in contracting muscle, actin does not move independently of myosin and that ATP hydrolysis is strongly coupled to the rotation of cross-bridges.

Chemiluminescent standards for quantitative comparison of two-dimensional electrophoresis western blots.

Chemiluminescent probes offer highly sensitive quantitative analyses of proteins blotted from electrophoretic gels onto a supporting matrix (e.g. nitrocellulose or polyvinylidene difluoride). Visualization of signals from probes involves the emission of light that is dependent on a number of variables (e.g. conjugated enzyme activity, antibody titer, hybridization efficiency, substrate concentration, chemical reactivity, temperature, etc.). Thus, it is important to be able to correct for these variations. For example, the exposure time of the blot to the detection medium (e.g., film or digital camera) is a critical variable in the final results. Several protein samples separated on a single blot (e.g. one-dimensional resolution) can be compared from the ratio of the individual proteins, but comparison of separate blots completed on different days requires a chemiluminescent standard. The situation is more complex when only one sample per gel/blot is used (i.e. two-dimensional electrophoresis (2-DE)). This paper describes a method for preparing agarose embedded standardized strips that contain dilutions of antigens that can be visualized with the corresponding probe antibody. This standardization strip can be produced from common laboratory supplies and provides a method to correct for alterations in chemiluminescent intensities from different 2-DE analysis. Several standardization strips can be produced simultaneously and then used during the electroblotting step of different blots on different days.

Identification of protein carbonyls after two-dimensional electrophoresis.

The oxidative modification of proteins plays a major role in a number of human diseases, but identity of the specific proteins that are most susceptible to oxidation has posed a difficult problem. Protein carbonyls are increased after oxidative stress, and after derivatization with 2,4-dinitrophenyl hydrazine (DNP) they can be detected by various analytical and immunological methods. Although high resolution two-dimensional electrophoresis (2-DE) can resolve virtually all proteins present in a cell or tissue it has been difficult to determine the oxidized proteins because the DNP-derivatization process alters the isoelectric points of proteins, and additional procedures must be utilized to remove reaction byproducts. These additional procedures can lead to loss of sample, and poor isoelectric resolution on immobilized pH gradient (IPG) strips. We have developed a method that allows the IPG strips to be derivatized with DNP directly following isoelectric focusing of the proteins. This method allows the visualization of oxidized proteins by 2-DE with high reproducibility.

Oral polymeric N-acetyl-D-glucosamine and osteoarthritis.

Many patients with arthritis are using alternative modes of therapy, including nutritional supplements, to treat their arthritis. Most patients never tell their doctors that they are taking alternative medications, and few doctors even ask about such activities. Over-the-counter supplements are expensive and consume large amounts of patients' healthcare dollars. Glucosamine has been widely touted as being an effective arthritis treatment. The authors designed and undertook a study to test the efficacy of a polymer of N-acetyl-D-glucosamine (NAG), or POLY-Nag, in a double-blind, placebo-controlled study in patients with osteoarthritis. Results indicate that POLY-Nag may be useful in treating patients with osteoarthritis.

Early bactericidal activity of amoxicillin in combination with clavulanic acid in patients with sputum smear-positive pulmonary tuberculosis.

The early bactericidal activity (EBA) of an antituberculosis agent is the rate of decrease in viable colony-forming units (CFU) per milliliter of sputum during the first 2 d of treatment of patients with previously untreated smear-positive pulmonary tuberculosis. The objective of this open randomized study was to evaluate the EBA of the combination of amoxicillin 3 g and clavulanic acid 750 mg. Ten patients with a mean age of 34 y and a mean weight of 56 kg received amoxicillin/clavulanic acid and 5 patients with a mean age of 34 y and a mean weight of 57 kg received no drug. In the patients receiving 1 dose of amoxicillin/clavulanic acid daily for 2 d the mean log10CFU/ml of sputum before treatment was 6.7402 (SD 0.539) and after 2 d of treatment 6.7046 (SD 0.609); the corresponding values in patients receiving no drug were 6.7823 (SD 0.563) and 6.7502 (SD 0.673), respectively. The EBA of 0.018 (SD 0.130) in patients receiving amoxicillin/clavulanic acid did not differ significantly from that of 0.016 (SD 0.069) in patients receiving no drug. It is unlikely that the combination of amoxicillin/clavulanic acid has an important place in the treatment of tuberculosis with the exception of those patients with multidrug-resistant tuberculosis who are otherwise therapeutically destitute.

Oxidized proteins in Alzheimer's plasma.

The levels of oxidatively modified proteins were examined in blood from Alzheimer's disease (AD) patients, non-AD controls, and AD relatives. Oxidative modification was measured by reacting the protein carbonyls with 2,4-dinitrophenyl hydrazine (DNPH). The total oxidized proteins were determined by HPLC, while specific protein oxidation was assessed from Western blots of electrophoretic gels using antibody to the DNP derivatives. Statistically significant elevations (P < 0.05) of total oxidized proteins were observed in both AD subjects and AD relatives when compared with non-AD controls. Moreover, a protein band (e.g., MW = 78-kDa) was uniquely oxidized in the plasma of AD subjects. Furthermore, this protein from AD subjects was more susceptible to in vitro oxidation. These data suggest that such oxidized proteins may be useful as biomarkers for the detection and evaluation of AD.

Reactive oxygen species: the unavoidable environmental insult?

Reactive oxygen species (ROS) are generated by a variety of sources from the environment (e.g., photo-oxidations and emissions) and normal cellular functions (e.g., mitochondrial metabolism and neutrophil activation). ROS include free radicals (e.g., superoxide and hydroxyl radicals), nonradical oxygen species (e.g., hydrogen peroxide and peroxynitrite) and reactive lipids and carbohydrates (e. g., ketoaldehydes, hydroxynonenal). Oxidative damage to DNA can occur by many routes including the oxidative modification of the nucleotide bases, sugars, or by forming crosslinks. Such modifications can lead to mutations, pathologies, cellular aging and death. Oxidation of proteins appears to play a causative role in many chronic diseases of aging including cataractogenesis, rheumatoid arthritis, and various neurodegenerative diseases including Alzheimer's Disease (AD). Our goal is to elucidate the mechanism(s) by which oxidative modification results in the disease. These studies have shown that (a) cells from old individuals are more susceptible to oxidative damage than cells from young donors; (b) oxidative protein modification is not random; (c) some of the damage can be prevented by antioxidants, but there is an age-dependent difference; and (d) an age-related impairment of recognition and destruction of modified proteins exists. It is believed that mechanistic insight into oxidative damage will allow prevention or intervention such that these insults are not inevitable. Our studies are also designed to identify the proteins which are most susceptible to ROS damage and to use these as potential biomarkers for the early diagnosis of diseases such as AD. For example, separation of proteins from cells or tissues on one- and two-dimensional gels followed by staining for both total protein and specifically oxidized residues (e.g., nitrotyrosine) may allow identification of biomarkers for AD.

A double stain for total and oxidized proteins from two-dimensional fingerprints.

Oxidative modification of proteins plays a major role in the etiology of aging and age-related diseases. For example, in Alzheimer's disease, although evidence points to oxidation of proteins as a causative factor in loss of cognitive abilities, it is not known which specific proteins of the brain are most susceptible to these modifications. Thus, it is of interest to identify the specific proteins which are susceptible to oxidation in vivo. Two-dimensional protein fingerprint methods offer the analytical potential for resolution of thousands of individual proteins from tissues, and the oxidized proteins can be visualized with immunological probes. Sensitive methods permit recovery and sufficient amino acid sequencing to identify these proteins. However, for such analyses it is essential to simultaneously analyze both protein content and level of oxidation. We have evaluated several approaches, identified the sources of artifacts and interferences, and developed a double-staining procedure that allows visualization and quantitation of total protein patterns as well as the specific oxidized proteins from two-dimensional protein fingerprints. The method has been applied to cells grown in culture and to tissue extracts from young and old animals.

Molecular wear and tear leads to terminal marking and the unstable isoforms of aging.

Our studies focus on the mechanisms of molecular wear and tear, terminal marking, protein degradation, and how these processes are altered with age. Molecular wear and tear directly links catalysis with postsynthetic terminal marking. For example, the binding of ligands and catalysis cause conformational changes that are transmitted from the catalytic center to the site of terminal marking and enhance the rates of specific covalent modifications, such as deamidation or oxidation. These oxidations or deamidations can introduce "KFERQ motifs" into proteins, which may permit them to be recognized and transported to the site(s) of complete degradation. Terminally marked proteins accumulate in aging cells and tissues and account for many of the health problems of the elderly. Two-dimensional protein fingerprinting coupled with immunostaining permits identification and characterization of these proteins. Free-radical traps or caloric restriction, which may prevent the formation or enhance the degradation of terminally marked proteins, may be useful in the prevention or treatment of age-associated health problems, including dementia.

Effect of active-site modification on the terminal marking deamidation of triosephosphate isomerase.

The conformational change which results from the opening and closing of the hinged lid over the catalytic center of triosephosphate isomerase is transmitted to the subunit interface of the dimer and eventually leads to the spontaneous, specific deamidation of Asn71. This is followed by the deamidation of Asn15 approximately 5 A away on the neighboring subunit and leads to destabilization of the protein and degradation. However, it has not been established whether this molecular wear and tear occurs via an intra-subunit or inter-subunit transmission of the conformational change. We have studied this first step in the terminal marking by reacting the active-site Glu165 with the substrate analog 3-chloroacetol phosphate (CAP) which immobilizes the hinged lid. Under mild deamidation conditions (pH 9.5, 24 h, 30 degrees C) the native homodimer readily deamidated. In contrast, the CAP-modified homodimers were resistant to deamidation. Heterodimers composed of one native subunit and one CAP subunit showed intermediate deamidation. When the native and CAP-labeled subunits were resolved by electrophoresis in urea gels, it was found that the unlabeled subunit had preferentially deamidated. These data coupled with molecular modeling considerations show that restricting movement of the hinged lid prevents deamidation of Asn71 on that subunit, but that the other subunit with the free hinged lid functions independently and still deamidates. Thus, the conformationally induced wear and tear appears to be largely intra-subunit. These observations clearly link catalysis with terminal marking of the protein and suggest a general mechanism for how other enzymes may wear out.

A prospective evaluation of children under the age of 5 years living in the same household as adults with recently diagnosed pulmonary tuberculosis.

SETTING: A South African suburb with a high tuberculosis incidence (> 800/100,000). OBJECTIVE: To determine the prevalence of tuberculosis infection and disease in children less than 5 years of age who were in close household contact with adults with pulmonary tuberculosis. DESIGN: Prospective clinical study. SUBJECTS: Children under 5 years of age (of whom > 98% had been BCG vaccinated in the neonatal period) in household contact with an adult with tuberculosis. INVESTIGATION: Clinical investigation, Mantoux skin testing, chest radiography, gastric aspirate culture for Mycobacterium tuberculosis. RESULTS: Of 155 children younger than 5 years in contact with 80 index cases (83% smear positive), 14% were infected and 34% diseased. Children aged under 2 years had more severe disease (endobronchial tuberculosis and bronchial compression). Of 154 household members aged over 5 years who were assessed, 17 had culture proven pulmonary tuberculosis (13 smear positive) and a further 16 were placed an antituberculosis treatment on the basis of radiological evidence. CONCLUSION: In a high tuberculosis incidence area evaluation of and chemoprophylaxis for childhood contacts of adults with pulmonary tuberculosis is a rewarding procedure. The detection of culture and smear positive pulmonary tuberculosis amongst adolescent and adult household contacts emphasizes the role of contact tracing in the detection of infectious cases of pulmonary tuberculosis and the prevention of the spread of tuberculosis.

Pilot study of oral polymeric N-acetyl-D-glucosamine as a potential treatment for patients with osteoarthritis.

Glucosamine and its derivatives, such as glucosamine sulfate and N-acetyl-D-glucosamine (NAG), have been shown to be effective in the treatment of patients with osteoarthritis. Unfortunately, the half-life of glucosamine in the blood is relatively short; therefore, a sustained-release form of the compound would be highly desirable. The purpose of this pilot study was to determine whether the polymeric form of NAG (POLY-Nag) could provide a longer-lasting oral source of NAG. Ten healthy subjects each ingested 1 g/d of either NAG or POLY-Nag for 3 days. After a 4-day washout period, each subject was crossed over to receive the other compound for 3 days. Serum samples were collected and analyzed using high-performance liquid chromatography. Results show that orally ingested NAG and POLY-Nag are absorbed, resulting in increased serum levels of NAG, and POLY-Nag appears to be at least as effective as NAG. Serum levels of NAG had decreased by 48 hours after cessation of ingestion of NAG or POLY-Nag but were still above baseline levels. Increases in serum glucosamine levels indicate that NAG and POLY-Nag are converted to glucosamine in vivo. In conclusion, POLY-Nag may provide a source of serum glucosamine for treatment of patients with osteoarthritis. Longer and more rigorous pharmaco-kinetic and clinical studies need to be done.

Delay in the diagnosis, notification and initiation of treatment and compliance in children with tuberculosis.

SETTING: The mortality and morbidity from childhood tuberculosis may be influenced by the delay from the time of first symptoms until the start of and compliance with treatment. OBJECTIVE: This study investigated these delay periods and the compliance with therapy in children with tuberculosis. DESIGN: During the study period there were 49 children with probable and 123 with confirmed pulmonary tuberculosis (WHO criteria). The mean period from first symptoms until presentation was 4.3 weeks, from presentation until notification 5 weeks and from notification until therapy 0.9 weeks. 16% of children notified as having tuberculosis never received therapy. Significantly fewer children in the urban squatter communities received therapy than in urban settled (P = 0.02), rural agricultural (P = 0.0001) and rural settled (P = 0.09) communities. 12% of children did not complete their therapy. CONCLUSION: The delay in presentation ('patient delay') was shorter than the delay in diagnosis ('doctor delay'). Failure to trace children and to complete therapy was particularly likely to occur in urban squatter communities. Easier access to health care facilities may shorten the 'patient delay' while greater awareness of tuberculosis and proper investigation of children may shorten the 'doctor delay'.

Value of the lateral chest radiograph in tuberculosis in children.

The value of the lateral chest radiograph, often considered a useful adjunct in the detection of hilar adenopathy, was evaluated in a prospective study of 449 children assessed for tuberculosis. Of these children 298 presented to the hospital with signs and symptoms suggestive of tuberculosis, while 151 were investigated in a regional clinic solely because they were in close contact with an adult household member on treatment for tuberculosis. Tuberculosis was confirmed by culture in 176 of the 449 children (39%). In 40 of these (23%) hilar adenopathy was visible on frontal and lateral view, in 19 of the 176 confirmed cases (11%) only on a frontal view and in 22 (13%) on a lateral view only. Probable tuberculosis was diagnosed in a further 140 of the 449 children (31%), and hilar adenopathy was visible on frontal and lateral views in 39 of these children (28%), on the frontal view only in 8 (6%) and on the lateral view only in 27 (19%). In the symptomatic children investigated in the hospital, and the asymptomatic children investigated in the clinic, hilar adenopathy was detected on the lateral chest radiograph only in 36 (12%) and 14 (9%) cases respectively. Lateral chest radiographs will considerably improve the accuracy of the diagnosis of childhood tuberculosis.