Gradient and GENERIC time evolution towards reduced dynamics.

Reduction of a mesoscopic dynamical theory to equilibrium thermodynamics brings to the latter theory the fundamental thermodynamic relation (i.e. entropy as a function of the thermodynamic state variables). The reduction is made by following the mesoscopic time evolution to its conclusion, i.e. to fixed points at which the time evolution ceases to continue. The approach to fixed points is driven by entropy, that, if evaluated at the fixed points, becomes the thermodynamic entropy. Since the fixed points are parametrized by the thermodynamic state variables (by constants of motion), the thermodynamic entropy arises as a function of the thermodynamic state variables and thus the final outcome of the reduction is the fundamental thermodynamic relation. This reduction process extends also to reductions in which the reduced theory still involves the time evolution (e.g. reduction of kinetic theory to hydrodynamics). The essence of the extension is the replacement of the mesoscopic time evolution of the state variables with the corresponding mesoscopic time evolution of the vector field (i.e. of the fluxes). The fixed point in this flux time evolution is the vector field generating the reduced mesoscopic time evolution. The flux-entropy driving the flux time evolution becomes, if evaluated at the fixed point, the flux fundamental thermodynamic relation in the reduced dynamical theory. We show that the flux-entropy is a potential related to the entropy production. This article is part of the theme issue 'Fundamental aspects of nonequilibrium thermodynamics'.

Basophils are not the key antigen-presenting cells in allergic patients.

BACKGROUND: Recent data obtained in mouse models have initiated a controversy whether basophils are the key antigen-presenting cells (APCs) in allergy. Here, we investigate whether basophils are of importance for the presentation of allergen and the induction of T cell proliferation in allergic patients. METHODS: T cells, basophils, and APCs depleted of basophils were purified from allergic patients. Co-culture systems based on purified major allergens were established to study allergen-specific T cell responses using proliferation assays. RESULTS: Only co-cultures of T cells with APCs depleted of basophils but not with basophils proliferated in response to allergen. Even addition of IL-3 to T cell-basophil co-cultures failed to induce allergen-specific T cell proliferation. CONCLUSIONS: Our data demonstrate by classical in vitro proliferation assays that basophils are not key antigen-presenting cells that promote T cell proliferation in secondary immune responses to allergen in allergic patients.

On the metal ion (Zn(2+), Cu(2+)) coordination with beta-amyloid peptide: DFT computational study.

As is widely accepted, the crucial event of the progress of Alzheimer's disease is the formation of extracellular neurotoxic amyloid plaques, consisting mainly of 40 or 42 amino acid residues, in the brain. Zinc and copper metal ions are involved in this process, since they enhance the amyloid beta aggregation and are incorporated into plaques. In this paper we have analyzed theoretically the energetics implied in the formation of some complexes of both cations, adopting a number of models that take into account various coordination environments. The aim was to determine which among the coordination patterns examined is the favoured one in order to give better insight in the controversy concerning the metal binding site of amyloid beta peptide.

Photooxidation technology for correlated light and electron microscopy.

The combination of the capabilities of light microscopical techniques with the power of resolution of electron microscopy along with technical advances has led to a gradual decline of the gap between classical light and electron microscopy. Among the correlative techniques using the synergistic opportunities, photooxidation methods have been established as valuable tools for visualizing cell structures at both light and electron microscopic level. Fluorescent dyes are used to oxidize the substrate diaminobenzidine, which in its oxidized state forms fine granular precipitates. Stained with osmium, the diaminobenzidine precipitates are well discernible in the electron microscope, thus labelling and defining the cellular structures, which at light microscopy level are recorded by fluorescent probes. The underlying photooxidation reaction is based on the excitation of free oxygen radicals that form upon illumination of fluorochromes; this is a central step in the procedure, which mainly influences the success of the method. This article summarizes basic steps of the technology and progresses, shows efforts and elaborated pathways, and focuses on methodical solutions as to the applicability of different fluorochromes, as well as conditions for fine structural localizations of the reaction products.

New organic FET-like photoactive device, experiments and DFT modeling.

We present the possible construction of an organic FET-like photoactive device in which source-drain current through a phthalocyanine ( H(2)Pc film is affected by a photo-induced dipolar field in a photoactive "gate" electrode. The influence of the dipolar electric field on charge transfer between H(2)Pc molecules is modeled by DFT quantum-chemical calculations on H(2)Pc dimers and tetramers.

Estimation of protease activity in soils at low temperatures by casein amendment and with substitution of buffer by demineralized water.

The aim of this work was to modify the method of Ladd and Buttler (1972), by substituting Tris-HCl buffer (pH 8.52) with demineralized water (DEMI H(2)O), in order to assess its suitability for measurement of casein-protease activity at pH levels close to those of real soil in H(2)O. Measurements were undertaken over a range of incubation temperatures from 3 to 49 degrees C. Testing was performed on one organic soil and two different mineral soils. The substitution of Tris-HCl buffer by DEMI H(2)O at 49 degrees C decreased casein-protease activity to 67.25% in mineral soil and to 53.76% in organic soil. With decreasing temperature casein-protease activity decreased the most in organic soil, i.e., 0.07% of original its value at 3 degrees C. The incubation period was extended to maximally 336 h at 3 degrees C to totally obtain >10.0% of L-tyrosine equivalents released at optimum or close to optimum temperature and pH conditions. The Q(10) values of casein-protease activity measured after substituting Tris-HCl buffer with DEMI H(2)O were unexpectedly high. Between the temperatures of 3 and 49 degrees C Q(10) ranged from 3.46 to 4.25, whereas between 3 and 25 degrees C Q(10) ranged from 6.78 to 11.08. Therefore, the modified method of Ladd and Buttler (1972) presented can be used for measurement of soil casein-protease activity under pH conditions close to that of real soil pH and at an averaged soil temperatures measured in the field. This modification makes possible an expression of soil casein-protease activity potential - when being combined with measurements of casein-protease activity under optimum or close to optimum temperature and pH conditions, if high concentration of casein is present.

Carotid endarterectomy after ischemic stroke--is there a justification for delayed surgery?

PURPOSE: To assess the relationship between outcome of carotid surgery and wait after ischemic stroke. METHODS: We retrospectively analysed data from patients undergoing carotid endarterectomy after ischemic stroke. We investigated the time interval between the event and endarterectomy in relation to surgical results and complications. RESULTS: Between January 2000 and December 2003, 104 patients were scheduled to undergo carotid endarterectomy after a recent stroke. Endarterectomy was performed within 6 h in seven patients (6.7%); within 4 weeks in 29 (27.9%); 4 weeks or more in 62 (59.6%) and six (5.8%) patients received no further therapy. Perioperative complications among patients treated within 4 weeks were 3.4% and were comparable to those treated after 4 weeks (4.8%). However, more than 12% of the patients awaiting operation experienced a new cerebrovascular event (ischemic stroke or carotid occlusion), most of them occurred in the 3rd or 4th week after the initial event. CONCLUSION: Our data indicates, that carotid endarterectomy can be performed with a comparable risk within a short delay after stroke. In addition severe cerebrovascular events occurring within the waiting period may be avoided.

Rectus femoris muscle flap donor-site morbidity.

Donor-site morbidity in four patients after reconstruction with free neurovascular rectus femoris muscle was examined through a series of strength tests in which the leg with rectus femoris muscle harvested was compared with the contralateral leg with an intact rectus femoris muscle. The tests were conducted with three testing devices: (1) the 'Con-Trex Leg-press' in which the force and power of right and left leg extensions at 0.2 and 0.4 m/s in a knee angle from 50 to 90 degrees were tested separately; (2) the isometric power tester, which enabled the unilateral evaluation of the isometric leg extension at three knee angles: 50, 70 and 90 degrees ; and (3) at the 'SP-Force Platforms' in which the patients performed a counter-movement jump where the amplitude of the ground reaction force, the parameters maximum force, and the jump height were calculated in order to compare the right and left leg during a single dynamic movement. Our results showed that the patients (with one exception) demonstrated a balanced relationship between the donor leg and the intact contralateral leg. The patient that primarily demonstrated a large strength deficit was retested 3 months later and showed, after an extensive rehabilitation and training program, an impressive increase in strength. The authors concluded that there is no significant limitation in the strength of the donor leg after removal of the rectus femoris muscle and consequently no significant functional donor-site morbidity. We believe that for the realisation of such results that the intraoperative linking of the vastus lateralis muscle with the vastus medialis muscle, especially in their lower third, and an extensive postoperative rehabilitation and training program are essential.

A variant of Cenani-Lenz syndactyly (CLS): review of the literature and attempt of classification.

An oligodactylous variant of Cenani-Lenz syndactyly and its surgical treatment is presented. The deformity is believed to be of autosomal recessive inheritance and caused by a disordered axial and longitudinal differentiation of the upper and lower extremities. The classical form is mainly characterised by a complete syndactyly of the hands. Malformations may also affect the bones of the forearm and, to a lesser extent, the lower limbs. We analysed clinical features and compared them with those previously described. According to our research of literature and our clinical findings there seem to exist two grossly different clinical phenotypes: spoon hand type and oligodactyly type. Typical constant clinical features such as carpal, metacarpal and digital synostoses, disorganisation of carpal bones, reduction of digital rays and syndactyly of toes are found in the reported cases. Inconstant features such as radio-ulnar synostosis, brachymesomelia, metatarsal synostoses and reduction of metatarsal rays may be present. In our case, successful bilateral digital ray individualisation and tendon transfers were performed to construct a grip function of the grossly deformed hands.

Decompression without anterior transposition: an effective minimally invasive technique for cubital tunnel syndrome.

Cubital tunnel syndrome represents the second most common compression neuropathy in the upper limb. There are three main surgical procedures to deal with this issue, namely simple decompression, medial epicondylectomy and anterior transposition. Nevertheless, optimal surgical treatment is still open to question. In the past three years we performed decompression of the nerve with or without external neurolysis and epineuriotomy on 52 patients (55 extremities). Preoperative diagnostic procedures included functional hand status, neurophysiological evaluation, X-ray of the elbow and neurosonography. Patients were then divided into three groups according to the staging criteria of Dellon. After an average follow-up of 13 months, the outcome was evaluated by complete examination of hand function, electrophysiological studies and interview with the patients. Postoperatively, two-point discrimination as well as strength improved significantly. Postoperative grip was 28.78 kg (79.8 % of the other hand), compared to 20.31 kg (58 % of the other hand) preoperatively (p = 0.000). Evaluation of each stage individually showed that the best functional outcome was achieved by the minimum-staged group with improvements in strength and sensory in all patients and total relief in two-thirds. In the severe-staged group, improvement could still be found in 75 % of the patients. Evaluation of conduction velocities showed highly significant improvements for both motor and sensory conduction velocities. In summary, simple decompression, if necessary modified with external neurolysis and epineuriotomy, showed high success rates in all stages. Decompression is a minimally invasive procedure, but very effective for mild as well as for severe cases and therefore the optimal treatment in cubital tunnel syndrome.

Vascular permeability and hyperpermeability in a murine adenocarcinoma after fractionated radiotherapy: an ultrastructural tracer study.

Large radiation doses cause postradiation vascular hyperpermeability by disrupting endothelia. The cumulative sequences of small doses (fractionated radiotherapy) standard in clinical practice cause it too, but not by endothelial disruption: the mechanisms are unknown. In this study, correlated fluorescent and ultrastructural localisation of a tracer revealed the architecture, fine structure and function of microvessels in mouse AT17 tumours, before and after 42 Gy fractionated radiation. Before irradiation, tumour vascular permeability lay in the normophysiological range defined by the gut and cerebral cortex. A double barrier regulated permeability: vesicular transport through the endothelial wall required approximately 2 h and then the basement membrane charge barrier trapped tracer for 2 h longer. Irradiation abolished the double barrier: tracer passed instantly through both endothelial wall and underlying basement membrane, forming diffusion haloes around microvessels within 2-5 min. Structurally, irradiated tumour microvessels were lined by a continuous and vital endothelium with closed interendothelial junctions; endothelial basement membranes were intact, though loosened. Irradiated endothelia exhibited extremely active membrane motility and intracellular vesicle trafficking. Radiation treatment raised vascular permeability by enhancing transendothelial transcytosis, and by altering the passive filter properties of the subendothelial basement membrane. This type of vascular hyperpermeability should be susceptible to pharmacological modulation.

Cryopreparation provides new insight into the effects of brefeldin A on the structure of the HepG2 Golgi apparatus.

High-pressure freezing and freeze-substitution were used to study Golgi ultrastructure and its brefeldin A-induced transformations in HepG2 human hepatoma cells. Cryoimmobilization arrested subcellular dynamics within milliseconds, thus considerably improving the temporal resolution in monitoring the very early effects of high brefeldin concentrations at the ultrastructural level (i.e., 20 microg/ml brefeldin applied for 35 s to 8 min). Moreover, this approach ruled out possible cumulative and/or synergistic effects of the drug and fixatives. Several findings differed from studies based on chemical fixation. In particular, Golgi breakdown did not proceed gradually but occurred in distinct steps. We found a conspicuous lag between the absence of nonclathrin coats on Golgi membranes after 30 s of brefeldin treatment and the disassembly of the stacks, which did not start until after 90 to 120 s. At this time, domains at the trans and cis faces separated from the stacks, starting tubulation and fragmentation. After 3-5 min the Golgi apparatus was completely replaced by loose meshworks of straight tubules of different sizes and staining properties; also frequent were bent tubules and vesicles forming glomerule-like structures. After 8 min all kinds of Golgi-derived structures had aggregated within huge clusters. The morphologically highly distinct structures found after brefeldin treatment could in part be correlated with particular Golgi domains in the control cells.

Tracing uptake of C3dg-conjugated antigen into B cells via complement receptor type 2 (CR2, CD21).

Electron microscopy was used to study the internalization and delivery of ligands for complement receptor type 2 (CR2, CD21) to endocytic compartments of B-lymphoblastoid Raji cells. Opsonized antigen was mimicked with purified C3dg conjugated to colloidal gold. C3dg-gold bound specifically to the cell surface in a time-dependent manner, and preincubation of the cells with a monoclonal antibody blocking the CR2 ligand-binding site completely inhibited any C3dg-gold binding. Notably, the binding of C3d-gold was confined to cell surface protrusions, eg, microvilli. C3dg-gold was apparently internalized through coated pits located at the bases of microvilli and could be traced to different compartments of the endocytic pathway. The morphologic characteristics and intracellular distribution of these multivesicular or multilaminar structures were compatible with those of compartments known to harbor major histocompatibility complex (MHC) class II molecules. Immunolabeling showed that the internalized C3dg-gold colocalized with MHC class II in these structures. These data provide the first ultrastructural evidence that complement-coated antigens are endocytosed by antigen-nonspecific B cells by CR2 and are delivered to the compartments in which peptide loading for antigen presentation occurs. They support the notion that CR2 may play a role in antigen presentation by B cells regardless of B-cell receptor specificity. (Blood. 2000;95:2617-2623)

Comparison of the construction of unmarked deletion mutations in Mycobacterium smegmatis, Mycobacterium bovis bacillus Calmette-Guérin, and Mycobacterium tuberculosis H37Rv by allelic exchange.

Until recently, genetic analysis of Mycobacterium tuberculosis, the causative agent of tuberculosis, was hindered by a lack of methods for gene disruptions and allelic exchange. Several groups have described different methods for disrupting genes marked with antibiotic resistance determinants in the slow-growing organisms Mycobacterium bovis bacillus Calmette-Guérin (BCG) and M. tuberculosis. In this study, we described the first report of using a mycobacterial suicidal plasmid bearing the counterselectable marker sacB for the allelic exchange of unmarked deletion mutations in the chromosomes of two substrains of M. bovis BCG and M. tuberculosis H37Rv. In addition, our comparison of the recombination frequencies in these two slow-growing species and that of the fast-growing organism Mycobacterium smegmatis suggests that the homologous recombination machinery of the three species is equally efficient. The mutants constructed here have deletions in the lysA gene, encoding meso-diaminopimelate decarboxylase, an enzyme catalyzing the last step in lysine biosynthesis. We observed striking differences in the lysine auxotrophic phenotypes of these three species of mycobacteria. The M. smegmatis mutant can grow on lysine-supplemented defined medium or complex rich medium, while the BCG mutants grow only on lysine-supplemented defined medium and are unable to form colonies on complex rich medium. The M. tuberculosis lysine auxotroph requires 25-fold more lysine on defined medium than do the other mutants and is dependent upon the detergent Tween 80. The mutants described in this work are potential vaccine candidates and can also be used for studies of cell wall biosynthesis and amino acid metabolism.

Reproductive termination in female Japanese monkeys: A comparative life history perspective.

This study explores the question of reproductive termination (loss of reproductive ability) in female Japanese macaques (Macaca fuscata) from the Arashiyama West (Texas) troop. We used a large sample of completed lives to identify reproductively terminated female Japanese macaques and to consider reproductive termination in Japanese macaques from a comparative life history perspective, which permits meaningful comparisons to be made with human female menopause. We classified a female as reproductively terminated if the time lag between last parturition and death exceeded two standard deviations of the female's own mean lifetime interbirth interval (Caro et al. [1995] Int. J. Primatol. 16:205-220). Seventy of the 95 females in the sample had at least 3 infants over their lifetime (the minimum required for the calculation of a mean and standard deviation), and thus were included in the analysis. Of these 70 females, 20 showed reproductive termination. Reproductively terminated females ranged in age from 14.5-32.7 years, although in females under age 25, reproductive termination was unlikely. The majority of females up to age 25 showed continued parturition. However, after age 25, reproductive termination was population-wide. Length of postreproductive life for reproductively terminated females varied from 0.07-4.4 years, with a mean of 2.08 years. Variation in length of postreproductive life was not related to the age at death of the female. While the occurrence of population-wide reproductive termination after 25 years does suggest similarities with human female menopause, the age at which this termination occurs is very late in the life span, and it was experienced by only 2.9% of the population. Female Japanese monkeys over age 25 are visibly aged and show outward signs of weakness and deterioration, quite unlike the healthy middle age of menopausal human females. Accordingly, as a life history characteristic, reproductive termination in Japanese macaques does not appear to coincide with menopause as experienced by human females.

Molecular characterization of an autoallergen, Hom s 1, identified by serum IgE from atopic dermatitis patients.

Atopy is a genetically determined disorder that affects 10%-20% of the population. Many symptoms of patients with atopy (allergic rhinitis, conjunctivitis, asthma, and anaphylaxis) result from events occurring after crosslinking of cell-bound IgE by per se innocuous environmental antigens. The frequently raised hypothesis that autosensitization can also be a pathogenetic factor in atopy, gained support by our recent demonstration of IgE antibodies against human proteins in atopic dermatitis patients. To unravel the molecular nature of IgE-defined autoantigens, we used serum IgE from atopic dermatitis patients to screen a human epithelial cDNA expression library. One of the cDNA-encoding IgE-reactive products contained 1501 bp of a 2274 bp open-reading frame finally identified by sequence analysis of two additional cDNA clones resulting from oligonucleotide screening. The IgE-defined autoantigen, designated Hom s 1, exhibited an almost complete sequence identity with a recently described antigen recognized by cytotoxic T cells of a squamous cell carcinoma patient. Purified recombinant Hom s 1 specifically bound IgE from patients with severe atopy. When used as immunogen in rabbits, recombinant Hom s 1 gave rise to an anti-serum that reacted with a cytoplasmic protein exhibiting a broad cellular and tissue reactivity (skin, lung >> gastrointestinal tract >> muscle, brain) and identified a 55 kDa protein in blotted serum IgE preparations. The attractive possibility remains that the Hom s 1-triggered IgE response contributes to the events resulting in allergic tissue inflammation. If so, the respective recombinant molecule may serve as a paradigmatic tool for the diagnosis and treatment of patients with "intrinsic" atopy.

Endocytic routes to the Golgi apparatus.

The endocytic routes of labelled lectins as well as cationic ferritin were studied in cells with a regulated secretion, i.e. pancreatic beta cells, and in constitutively secreting cells, i.e. fibroblasts and HepG2 hepatoma cells, paying particular attention to routes into the Golgi apparatus. Considerable amounts of internalised molecules were taken up into the trans Golgi network (TGN) and into Golgi subcompartments in all three cell types as well as in secretory granules of the pancreatic beta cells. The internalised materials did not pass rapidly the TGN and Golgi stacks, but were still present hours after internalisation, being then particularly concentrated in TGN-elements and in the transmost Golgi cisterna. Endocytosed materials reached forming secretory granules present in the TGN. Further, direct fusion between endocytotic vesicles and mature secretory granules was observed. Golgi subcompartments as well as endocytic TGN containing endocytosed materials were in close apposition to specialised regions of the endoplasmic reticulum. The Golgi apparatus including its parts containing endocytosed materials were transformed into a tubular reticulum upon treatment with the fungal metabolite Brefeldin A. Rarely, internalised material was observed in the lumen of the endoplasmic reticulum, thus providing evidence for an endocytic plasma membrane to endoplasmic reticulum route.

The possible role of the immune system in Alzheimer's disease.

Currently, there is little doubt that the immune system plays a role in the neurodegenerative process in Alzheimer's disease (AD). Inflammatory proteins such as complement components, enzymes, eicosanoids, and cytokines are found in association with cerebral amyloid plaques and may exacerbate the fundamental pathology of AD, by stimulating Amyloid beta (A beta) production, supporting its aggregation and increasing its cytotoxicity. Activated microglia and astrocytes are the main source of these proteins, and A beta may trigger their release. Interestingly, there are also indications that the immune system may play a protective role against the development of AD. Microglial cells have been shown to degrade A beta, and recent evidence suggests that autoreactive A beta-specific T cells may be relevant to the elimination of the peptide. This mechanism seems, however, impaired in the majority of patients with AD. The immune system seems thus to represent a natural line of defense against the accumulation of dangerous amyloidogenic substances. Impairment of this specific immunological defense mechanism and the failure to eliminate a toxic metabolite can be the basis for a chronic nonspecific inflammatory process in the brain, as described above. AD is a good example how an immune response initially aiming at maintaining the integrity of the body may fail and consequently lead to tissue destruction and neuronal loss.

Irradiation induces G2/M cell cycle arrest and apoptosis in p53-deficient lymphoblastic leukemia cells without affecting Bcl-2 and Bax expression.

The tumor suppressor p53 has been implicated in gamma irradiation-induced apoptosis. To investigate possible consequences of wild-type p53 loss in leukemia, we studied the effect of a single dose of gamma irradiation upon p53-deficient human T-ALL (acute lymphoblastic leukemia) CCRF - CEM cells. Exposure to 3 - 96 Gy caused p53-independent cell death in a dose and time-dependent fashion. By electron microscopic and other criteria, this cell death was classified as apoptosis. At low to intermediate levels of irradiation, apoptosis was preceded by accumulation of cells in the G2/M phase of the cell division cycle. Expression of Bcl-2 and Bax were not detectably altered after irradiation. Expression of the temperature sensitive mouse p53 V135 mutant induced apoptosis on its own but only slightly increased the sensitivity of CCRF - CEM cells to gamma irradiation. Thus, in these, and perhaps other leukemia cells, a p53- and Bcl-2/Bax-independent mechanism is operative that efficiently senses irradiation effects and translates this signal into arrest in the G2/M phase of the cell cycle and subsequent apoptosis.

Cloning of the dapB gene, encoding dihydrodipicolinate reductase, from Mycobacterium tuberculosis.

Diaminopimelate (DAP) is used by bacteria for the synthesis of lysine. In many species of bacteria, including mycobacteria, DAP is also used for peptidoglycan biosynthesis. In this report we describe the cloning of the dapB gene encoding dihydrodipicolinate reductase (DHPR), which catalyzes a key branch point reaction in the bacterial DAP biosynthetic pathway, from Mycobacterium tuberculosis. Analyses of the DapB proteins from different bacterial species suggest that two different classes of DHPR enzymes may exist in bacteria.