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PubMedDate not supplied

Immunoelectron microscopy of Giardia lamblia cytoskeleton using antibody to acetylated alpha-tubulin.

Giardia lamblia trophozoites contain acetylated alpha-tubulin but lack detectable levels of tyrosinolated alpha-tubulin, as demonstrated in immunoblots with monoclonal antibodies specific for these tubulin forms. By immunofluorescence microscopy, acetylated alpha-tubulin is localized in axonemes, median bodies and in the adhesive disk. Post-embeddment immunogold labeling of thin sections of cells was used to evaluate acetylation at the level of individual microtubules by electron microscopy. Cells were fixed with glutaraldehyde and embedded in the acrylic resin LR Gold. Results indicate all microtubules in adhesive disk, axonemes, basal bodies, funis and the median bodies contain acetylated alpha-tubulin. Unlike immunofluorescence labeling, all microtubules of the adhesive disk and the funis could be gold labeled. No nonspecific labeling of the cytoplasm or of structures other than microtubules was observed. Acetylated microtubules in G. lamblia do not appear to be a subset of microtubules and acetylation appears uniform along the entire length of individual microtubules. Acetylation and the tyrosinolation state of microtubules in Giardia are discussed in the context of microtubule stability and crosslinked features of the cytoskeleton.

Acetylation↗

PubMed1986

Isolation and Characterization of an Fe(III)-Chelating Compound Produced by Pseudomonas syringae.

The phytopathogenic bacterium Pseudomonas syringae produces a fluorescent pigment when it is grown in iron-deficient media. This pigment forms a very stable Fe(III) complex that was purified in this form by using a novel procedure based on ultrafiltration and column chromatography. The Fe(III) complex has a molecular weight of 1,100 and contains 1 mol of Fe(III). The pigment is composed of an amino acid moiety with three threonines, three serines, one lysine, delta-N-hydroxyornithine, and a quinoline-type fluorescent chromophore. These features and its stability constant (in the range of 10) suggest that the fluorescent pigment of P. syringae is related to the siderophores produced by another Pseudomonas species.

Journal Article↗

PubMed2000

Tectal gliomas: natural history of an indolent lesion in pediatric patients.

The mesencephalic tectal glioma is a distinctive form of brain stem glioma with an unusually benign clinical course. Periaqueductal location, lack of contrast enhancement, and long periods of stability are classic features. The clinical management of these lesions, especially at the time of radiographic enlargement varies widely in the published literature. It is unclear whether these progressive lesions need to be treated. Accordingly, clinical and radiologic features of 7 patients were reviewed, with attention to the clinical course of the disease after radiologic enlargement. The age at diagnosis ranged from 3.3 to 16.6 years. Six of 7 had MRI tumor enlargement beginning 0.3-5.7 years after initial diagnosis. One of these 6 patients had radiographic progression coupled with a new clinical symptom which was treated with stereotactic radiation therapy. The remaining 5 patients with MRI progression and normal neurological exams were not treated and remain free of new neurologic deficits 1.8-6.9 years after the first radiographic tumor enlargement. The results suggest that pediatric tectal gliomas are a very low-grade lesion. Conservative management in the absence of new clinical symptoms could be argued, reserving radiotherapy or chemotherapy for clinical progression.

Adolescent↗

PubMed2006

Receptive field structures for recognition.

Localized operators, like Gabor wavelets and difference-of-gaussian filters, are considered useful tools for image representation. This is due to their ability to form a sparse code that can serve as a basis set for high-fidelity reconstruction of natural images. However, for many visual tasks, the more appropriate criterion of representational efficacy is recognition rather than reconstruction. It is unclear whether simple local features provide the stability necessary to subserve robust recognition of complex objects. In this article, we search the space of two-lobed differential operators for those that constitute a good representational code under recognition and discrimination criteria. We find that a novel operator, which we call the dissociated dipole, displays useful properties in this regard. We describe simple computational experiments to assess the merits of such dipoles relative to the more traditional local operators. The results suggest that nonlocal operators constitute a vocabulary that is stable across a range of image transformations.

Action Potentials↗

PubMed2006

Mutation causing self-aggregation in human gammaC-crystallin leading to congenital cataract.

PURPOSE: Many forms of congenital hereditary cataract are associated with mutations in the crystallin genes. The authors focus attention on congenital lamellar cataract, which is associated with the R168W mutation in gammaC-crystallin, and congenital zonular pulverulent cataract, which is associated with a 5-bp insertion in the gammaC-crystallin gene. METHODS: To understand the molecular phenotypes-i.e., the functional defects that have occurred in the mutant gammaC-crystallin molecule in two cases described-the authors cloned, expressed, isolated, and compared the solution state structural features of these mutants with those of normal (wild-type) gammaC-crystallin. Structural models of the wild-type and mutant have been generated using comparative modeling. Circular dichroism and fluorescence spectroscopic methods were used to determine the conformation of the proteins, and temperature dependent self-aggregation was used to observe the quaternary structural features. The structural stability of the proteins was monitored with the use of chemical and thermal denaturation. RESULTS: The authors found that the 5-bp insertion led to a loss of secondary and tertiary structures of the molecule and to an enhanced tendency of self-aggregation into light-scattering particles, offering a possible factor in lens opacification. The R168W mutant, on the other hand, was remarkably similar to the wild-type molecule in its conformation and structural stability, but it differed in its ability to aggregate and scatter light. CONCLUSIONS: These results support the idea that unfolding or structural destabilization is not always necessary for crystallin-associated cataractogenesis.

Cataract↗

PubMedDate not supplied

Skiing orthosis for recurrent shoulder dislocation.

A functional orthosis has been designed and implemented with the Alpine skier in mind. The unique features include a stabilizing pelvic band and dynamic abduction and external rotation check straps. A case report is presented.

Adolescent↗

PubMed2005

Simplified screening for the detection of soluble fusion constructs expressed in E. coli using a modular set of vectors.

BACKGROUND: The solubility of recombinant proteins expressed in bacteria is often disappointingly low. Several strategies have been developed to improve the yield and one of the most common strategies is the fusion of the target protein with a suitable partner. Despite several reports on the successful use of each of these carriers to increase the solubility of some recombinant proteins, none of them was always successful and a combinatorial approach seems more efficient to identify the optimal combination for a specific protein. Therefore, the efficiency of an expression system critically depends on the speed in the identification of the optimal combination for the suitable fusion candidate in a screening process. This paper describes a set of expression vectors (pETM) designed for rapid subcloning, expression and subsequent purification using immobilized metal affinity chromatography (IMAC). RESULTS: A single PCR product of two Yellow Fluorescent Proteins (EYFPs) was cloned into 18 vectors comprising identical restriction sites and varying fusion partners as well as differing protease recognition sites. After a small-scale expression, the yields of the different constructs were compared using a Coomassie stained SDS-polyacrylamide gel and the results of this preliminary screening were then confirmed by large-scale purification. The yields were calculated and the stability of the different constructs determined using three independent conditions. The results indicated a significant correlation between the length and composition of non-native amino acid tails and stability. Furthermore, the buffer specificity of TEV and 3C proteases was tested using fusion proteins differing only in their protease recognition sequence, and a His-GST-EYFP construct was employed to compare the efficiency of the two alternative affinity purification methods. CONCLUSION: The experiments showed that the set of pETM vectors could be used for the rapid production of a large array of different constructs with specific yield, stability, and cleavage features. Their comparison allowed the identification of the optimal constructs to use for the large-scale expression. We expect that the approach outlined in this paper, i.e. the possibility to obtain in parallel fusion products of the target protein with different partners for a preliminary evaluation, would be highly beneficial for all them who are interested in the rapid identification of the optimal conditions for protein expression.

Journal Article↗

PubMed2005

Divergence, recombination and retention of functionality during protein evolution.

We have only a vague idea of precisely how protein sequences evolve in the context of protein structure and function. This is primarily because structural and functional contexts are not easily predictable from the primary sequence, and evaluating patterns of evolution at individual residue positions is also difficult. As a result of increasing biodiversity in genomics studies, progress is being made in detecting context-dependent variation in substitution processes, but it remains unclear exactly what context-dependent patterns we should be looking for. To address this, we have been simulating protein evolution in the context of structure and function using lattice models of proteins and ligands (or substrates). These simulations include thermodynamic features of protein stability and population dynamics. We refer to this approach as 'ab initio evolution' to emphasise the fact that the equilibrium details of fitness distributions arise from the physical principles of the system and not from any preconceived notions or arbitrary mathematical distributions. Here, we present results on the retention of functionality in homologous recombinants following population divergence. A central result is that protein structure characteristics can strongly influence recombinant functionality. Exceptional structures with many sequence options evolve quickly and tend to retain functionality--even in highly diverged recombinants. By contrast, the more common structures with fewer sequence options evolve more slowly, but the fitness of recombinants drops off rapidly as homologous proteins diverge. These results have implications for understanding viral evolution, speciation and directed evolutionary experiments. Our analysis of the divergence process can also guide improved methods for accurately approximating folding probabilities in more complex but realistic systems.

Evolution, Molecular↗

PubMed1993

Amyloid destructive spondyloarthropathy causing cord compression: related to chronic renal failure and dialysis.

Destructive spondyloarthropathy is a recently recognized disease that has not been reported in the neurosurgical literature. It is associated with spinal amyloid deposition in long-term renal failure and dialysis, and it occurs increasingly as the number of dialysis patients and their survival times increase. Clinically, there is a multisegmental and often rapidly progressive radiculomyelopathy that may require emergency stabilization. The radiological features are disc space narrowing with erosion of vertebral end plates and subarticular cysts. The pathological features include deposition of amyloid, which stains with Congo Red and antibodies to beta-2-microglobulin. We present two cases with clinical, radiological, and pathological features and a review of the literature.

Aged↗

PubMed2006

Promyelocytic leukemia nuclear bodies are predetermined processing sites for damaged DNA.

The promyelocytic leukemia protein (PML) participates in several cellular functions, including transcriptional regulation, apoptosis and maintenance of genomic stability. A key feature of this protein is its ability to induce the assembly of nuclear compartments termed PML-nuclear bodies (PML-NBs). Here we show that these nuclear structures recruit single-stranded DNA (ssDNA) molecules in response to exogenous DNA damage. ssDNA was readily detected in PML-NBs within 1 hour following exposure of cells to UV light. Confocal real-time imaging of cells expressing YFP-tagged PML did not reveal de novo formation of new PML-NBs following UV-irradiation, which shows that ssDNA focus formation occurred within pre-existing PML-NBs. Moreover, siRNA-mediated depletion of PML prevented ssDNA focus formation and sensitized cells to UV-induced apoptosis. PML-dependent ssDNA focus formation was found to be particularly efficient during S-phase of the cell cycle, and PML-depleted cells became retarded in S-phase upon growth in the presence of etoposide. In addition, we found that caffeine and the poly(ADP-ribose) polymerase (PARP) inhibitor NU1027 enhanced UV-induced recruitment of ssDNA to PML-NBs. Together, our results show that PML-NBs have the capacity to accommodate DNA metabolic activities that are associated with processing of damaged DNA.

Apoptosis↗

PubMed2006

Error analysis for rotating active Stokes-Mueller imaging polarimeters.

Retrieval of Mueller matrices from intensity measurements is a noise-sensitive process. In addition, the choice of the method used for extracting Mueller matrix elements greatly influences the precision of the final results. Among available procedures, three have been tested and their robustness analyzed by adding Gaussian noise to computer synthesized data and have been verified by comparing experimental data. As expected, the methods based on classical matrix inversion reach their noise reduction limit even if more data are considered. In contrast, the discrete Fourier transform approach features a remarkable stability. The identified reason is that the retrieval process corresponds to filtering with a periodic kernel.

Journal Article↗

PubMed2002

Calcium-alginate gel bead cross-linked with gelatin as microcarrier for anchorage-dependent cell culture.

Valuable products obtainedfrom the cultivation of anchorage-dependent mammalian cells require large-scale processes to obtain commercially useful quantities. It is generally accepted that suspension culture is the ideal mode of operation. Because anchorage-dependent cells need surfaces to be able to attach and spread, the incorporation of microcarriers to suspension culture is indispensable. Since the dextran-based microcarrier wasfirst introduced, many different types of microcarriers have been developed and commercialized. In this study, alginate-based microcarriers were made in the following order: (i) calcium-alginate gel beads prepared by dropping a blend of sodium alginate and propylene glycol alginate (PGA) into calcium chloride solution, (ii) the PGA section of gel beads cross-linked with gelatin in alkaline solution (i.e., via the transacylation reaction between the ester group of PGA and amino group of gelatin), and (iii) gelatin membrane around the beads further cross-linked by glutaraldehyde. The glutaraldehyde-treated gelatintransacylated PGA/alginate microcarrier showed superior features in high stability under phosphate-containing solution, density close to that of culture medium, and transparency. Moreover, the Chinese hamster ovary CHO-KI and amphotropic retrovirus producer PA317 cells cultivated on the newly synthesized microcarriers exhibited similar growth kinetics of these two types of cell lines cultured on commercial polystyrene microcarriers. However, cell morphology was easily monitored on the transparent microcarriers made in this study.

Alginates↗

PubMed2003

Interactions of VIP, secretin and PACAP(1-38) with phospholipids: a biological paradox revisited.

Vasoactive intestinal peptide (VIP), secretin and pituitary adenylate cyclase-activating peptide(1-38)(PACAP(1-38)) are widely distributed amphipathic mammalian neuropeptides that exert diverse biological effects in target tissues located distant from their site of release. However, the half-life of exogenously-administered VIP, secretin and PACAP(1-38) in the bloodstream is relatively short (minutes) due to rapid degradation and inactivation. This seemingly paradoxical behavior suggests the presence of an innate system(s) that protects the peptides from degradation in vivo. To this end, VIP, secretin and PACAP(1-38) express distinct biophysical properties that once released may protect them from degradation in biological fluids. They self-aggregate at low (nanomolar) concentrations and interact avidly with biomimetic phospholipid monolayers and bilayers at physiological concentrations. The latter evokes conformational transition of the VIP, secretin and PACAP(1-38) molecules from predominantly random coil in aqueous solution to alpha-helix, the preferred peptide conformation for receptor interaction, in phospholipids. These features increase peptide stability and amplify bioactivity in vivo. Collectively, these data suggest the presence of an endogenous targeted delivery platform for VIP, secretin and PACAP(1-38). This innate system may constitute a novel molecular recognition paradigm that could also apply to other amphipathic neuropeptides. Importantly, the distinct behavior of VIP, secretin and PACAP(1-38) in the presence of phospholipids could be exploited to develop novel, long-acting therapeutic formulations of these peptides.

Animals↗

PubMed1999

Histopathology of a well-functioning hydroxyapatite-coated femoral prosthesis after 52 months.

A fully hydroxyapatite-coated femoral implant was retrieved during autopsy. This component, provided with a bipolar femoral head, had been inserted for a displaced fracture of the femoral neck 52 months before. Osseointegration of the implant was evident, without any formation of fibrous tissue :39.9% of the perimeter of the prosthesis at the level of its proximal third was interfaced with bone (62.8% at the mid-third and 65.2% at the distal third). Remodeling of bone had ensued. Deposition of bone was most prominent in the calcar zone, along the medial and lateral aspects and around the tip. Proximally, cortical porosity was found to be increased by 73%, whereas medullary bone porosity was increased by a factor of 2. Cell-mediated resorption of the coating was systematically present in these bone remodeling areas. The average thickness of the coating was respectively 10.8, 50.2 and 151.2 microns in the proximal, mid- and distal thirds of the implant. Formation of new bone was often coupled with resorption. No debris from the coating was found in the joint tissues or in the articulating surface of the polyethylene insert. These overall histopathological features support mechanical stability of the implant and active remodelling of bone along with focal removal of HA coating associated with osteoclastic activity. No side effects from coating degradation could be demonstrated.

Aged↗

PubMed2000

[Point amino acid substitutions in Ca2+-binding centers of recoverin. III. Mutant with the fourth reconstructed Ca2+-binding site].

Unlike wild type recoverin with only two (the second and the third) functioning Ca(2+)-binding sites out of four potential ones, the +EF4 mutant contains a third active Ca(2+)-binding site. This site was reconstructed from the fourth potential Ca(2+)-binding domain by the introduction of several amino acid substitutions in it by site-directed mutagenesis. The effect of these mutations in the fourth potential Ca(2+)-binding site of myristoylated recoverin on the structural features and conformational stability of the protein was studied by fluorimetry and circular dichroism. The apoform of the resulting mutant (free of Ca2+ ions) was shown to have a higher calcium capacity, significantly lower thermal stability, and noticeably different secondary and tertiary structures as compared with the apoform of wild type recoverin.

Amino Acid Substitution↗

PubMed1975

[Morphological aspects of the functional stability of the kidney].

Specific features of the internal architectonics of the secondary kidney have been analyzed from standpoints of main propositions of cybernetics. The high stability of the organ as a biological system is determined by a great number of non-equivalent elements both in the composition of the nephron in the level of coalition of nephrons and in other subsystems. It promotes 1) preservation of a part of neurons under extreme effects, 2) duplication, 3) creation of optimal variants of active functioning coalitions of nephrons, 4) maintenance of optimal regimen of work of the nephron and kidney as a whole. Specialization of cells, their ability to regenerate, control in many levels also contribute to stability. It is supposed that non-equivalence of the elements of subsystems in ontogenesis is sustained by their asynchronous development, while in phylogenesis--by intratissue divergence.

Animals↗

PubMedDate not supplied

[Use of a bilateral stabilographic platform in the diagnosis and rehabilitation of movement disorders at the clinic of nervous system diseases].

The paper presents the results of the study whose aims were to reveal the specific features of vertical posture maintenance in patients with various motor disorders by using a computer stabilographic complex with a bilateral platform, to draw up computer rehabilitative programmes that take into account the detected features of posture stability disorders in these groups of patients, to evaluate the efficiency of use of programmes drawn up to train vertical posture stability in the rehabilitation of patients with motor disorders of various origin.

Adult↗

PubMed2001

Cholesterol lowering and the vessel wall: new insights and future perspectives.

The basis for most acute coronary events is either rupture or fissuring of unstable atherosclerotic plaques with subsequent thrombosis leading to coronary artery occlusion. The development of atherosclerotic plaques takes several decades, but the mechanical features determining its stability and the risk of rupture can change very rapidly depending on a number of internal factors. Unstable plaques have a large lipid core, a thin overlying fibrous cap and an abundance of inflammatory cells. The most important factor determining the plaque stability is the plasma level of atherogenic LDL particles. Increased levels of these particles cause endothelial dysfunction with impaired vasodilatation capacity and prevalence of vasoconstriction, maintain inflammatory infiltration of the plaque, impair the strength of the fibrous cap and facilitate aggregation and coagulation. Effective lowering of plasma cholesterol by pharmacological and non-pharmacological means can revert most of these processes and increase the plaque's mechanical stability within several hours to days. Lipid lowering therapy can therefore decrease the risk of acute coronary events within a very short space of time. Thus a radical decrease in lipid levels, along with modification of other risk factors, may become the cornerstone for treatment of acute coronary syndromes, in addition to being an effective treatment in primary and secondary prevention of coronary heart disease (CHD).

Anticholesteremic Agents↗