Negative SPR Signals during Low Molecular Weight Analyte Recognition.

Surface plasmon resonance (SPR) is a powerful technique for studying biomolecular interactions mainly due to its sensitivity and real-time and label free advantages. While SPR signals are usually positive, only a few studies have reported sensorgrams with negative signals. The aim of the present work is to investigate and to explain the observation of negative SPR signals with the hypothesis that it reflects major changes in ligand conformation resulting from target binding. We demonstrated that these negative unconventional signals were due to the negative complex (ligand/analyte) refractive index increment (RII) deviation from the sum of the RII of the individual entities which counterbalanced the theoretical increase of the signal triggered by the target recognition and the ligand folding. We also found that the conformation change of biomolecules can induce a negative or a positive complex RII deviation depending on its sequence and immobilization mode.

Influence of the SPR Experimental Conditions on the G-Quadruplex DNA Recognition by Porphyrin Derivatives.

Surface plasmon resonance (SPR) is a powerful technique to study the interactions of ligands with analytes and therefore a number of biosensor surfaces and injection methods have been developed so far. However, many experimental parameters can affect the interactions and consequently the affinity measurements. In particular, the interactions of positively charged analytes (often used for anionic nucleic acids targets) can be influenced by the sensing surfaces (e.g., negatively charged), leading to significant nonspecific interactions as well as regeneration problems. The aim of the present work is to investigate the effect of different parameters, including ionic strength, SPR biosensor (i.e., nature of the surfaces), and the injection method on the recognition of porphyrin G-quadruplex ligands. We demonstrate that the injection method does not influence the affinity whereas the ionic strength and the nature of the surface impact the recognition properties of the porphyrin for the G-quadruplex DNA. We also found that self-assembled monolayer coating surface presents many advantages in comparison with carboxymethylated dextran surface for SPR studies of G-quadruplex DNA/ligand interactions: (i) the electrostatic interaction with charged analytes is less important, (ii) its structure/composition is less sensitive to the ionic concentration and less prone to unspecific adsorption, (iii) it is easily homemade, and (iv) the cost is approximately 10 times cheaper.

A one-dimensional moving-boundary model for tubulin-driven axonal growth.

A one-dimensional continuum-mechanical model of axonal elongation due to assembly of tubulin dimers in the growth cone is presented. The conservation of mass leads to a coupled system of three differential equations. A partial differential equation models the dynamic and the spatial behaviour of the concentration of tubulin that is transported along the axon from the soma to the growth cone. Two ordinary differential equations describe the time-variation of the concentration of free tubulin in the growth cone and the speed of elongation. All steady-state solutions of the model are categorized. Given a set of the biological parameter values, it is shown how one easily can infer whether there exist zero, one or two steady-state solutions and directly determine the possible steady-state lengths of the axon. Explicit expressions are given for each stationary concentration distribution. It is thereby easy to examine the influence of each biological parameter on a steady state. Numerical simulations indicate that when there exist two steady states, the one with shorter axon length is unstable and the longer is stable. Another result is that, for nominal parameter values extracted from the literature, in a large portion of a fully grown axon the concentration of free tubulin is lower than both concentrations in the soma and in the growth cone.

Abnormal axonal guidance and brain anatomy in mouse mutants for the cell recognition molecules close homolog of L1 and NgCAM-related cell adhesion molecule.

Cell recognition molecules of the L1 family serve important functions in the developing and the mature nervous system. Mutations in genes encoding the L1 family members close homolog of L1 (CHL1) and NgCAM-related cell adhesion molecule (NrCAM) have been found to alter connectivity and morphology of several brain regions. In order to emphasize similarities and differences of these two structurally related molecules, null mutants for CHL1 and NrCAM were directly compared with respect to axonal guidance in the hippocampus and the olfactory bulb and the sizes of the ventricular system and the cerebellar vermis using a combined structural magnetic resonance imaging (MRI) and histological approach. The results demonstrate that the absence of CHL1 leads to aberrant hippocampal mossy fiber projections whereas in both mutants, CHL1 and NrCAM, the guidance of the olfactory nerve projections is disturbed. Both mutations also alter the size of the ventricular system and the vermis with a specific profile of changes and partially opposite effects in each of the mutants. CHL1/NrCAM double-mutant mice do not show any enhancement of the single mutant's phenotype but balance the opposing effects on the ventricular system. In summary, the results show that CHL1 and NrCAM both affect axonal guidance and the anatomy of the ventricular system and the cerebellar vermis but act differently on these processes.

Simulation of nitrification of municipal wastewater in a Moving Bed biofilm process: a bottom-up approach based on a 2D-continuum model for growth and detachment.

This paper presents a complete mathematical model of a Moving Bed biofilm process for waste-water treatment, in particular for the nitrification process. The model is based on a bottom up approach adopting a multidimensional model for the biofilm growth and metabolism and a global mass balance model for the whole reactor. The model shows that oxygen is limiting the amount of biomass involved in the nitrification process. Furthermore, it suggests the existence of an optimal amount biomass for an optimal reactor turnover rate. Studies of two specific new suspended carriers show that the model output is dependable on the geometry of the carrier, and to a satisfactory extent agreeable with measurements.

A new mathematical model for chemotactic bacterial colony growth.

A new continuum model for the growth of a single species biofilm is proposed. The geometry of the biofilm is described by the interface between the biomass and the surrounding liquid. Nutrient transport is given by the solution of a semi-linear Poisson equation. In this model we study the morphology of a chemotactic bacterial colony, which grows in the direction of increasing nutrient concentration. Numerical simulations using the level set method and finite difference schemes are presented. The results show rich heterogeneous morphology.

Antidepressants enhance glucocorticoid receptor function in vitro by modulating the membrane steroid transporters.

1. Previous data demonstrate that the tricyclic antidepressant, desipramine, induces glucocorticoid receptor (GR) translocation from the cytoplasm to the nucleus in L929 cells and increases dexamethasone-induced GR-mediated gene transcription in L929 cells stably transfected with the mouse mammary tumour virus-chloramphenicol acetyltransferase (MMTV-CAT) reporter gene (LMCAT cells) (Pariante et al., 1997). 2. To extend these findings, the present study has investigated the effects of 24 h coincubation of LMCAT cells with dexamethasone and amitriptyline, clomipramine, paroxetine, citalopram or fluoxetine. 3. All antidepressants, except fluoxetine, enhanced GR-mediated gene transcription, with clomipramine having the greatest effect (10 fold increase). Twenty-four hours coincubation of cells with desipramine, clomipramine or paroxetine, also enhanced GR function in the presence of cortisol, but not of corticosterone. 4. It is proposed that these effects are due to the antidepressants inhibiting the L929 membrane steroid transporter, which actively extrudes dexamethasone and cortisol from the cell, but not corticosterone. This is further confirmed by the fact that clomipramine failed to enhance GR-mediated gene transcription in the presence of dexamethasone when the membrane steroid transporter was blocked by verapamil. 5. The membrane steroid transporters that regulate access of glucocorticoids to the brain in vivo, like the multiple drug resistance p-glycoprotein, could be a fundamental target for antidepressant action.

Automated interpretation of ventilation-perfusion lung scintigrams for the diagnosis of pulmonary embolism using artificial neural networks.

The purpose of this study was to develop a completely automated method for the interpretation of ventilation-perfusion (V-P) lung scintigrams used in the diagnosis of pulmonary embolism. An artificial neural network was trained for the diagnosis of pulmonary embolism using 18 automatically obtained features from each set of V-P scintigrams. The techniques used to process the images included their alignment to templates, the construction of quotient images based on the ventilation and perfusion images, and the calculation of measures describing V-P mismatches in the quotient images. The templates represented lungs of normal size and shape without any pathological changes. Images that could not be properly aligned to the templates were detected and excluded automatically. After exclusion of those V-P scintigrams not properly aligned to the templates, 478 V-P scintigrams remained in a training group of consecutive patients with suspected pulmonary embolism, and a further 87 V-P scintigrams formed a separate test group comprising patients who had undergone pulmonary angiography. The performance of the neural network, measured as the area under the receiver operating characteristic curve, was 0.87 (95% confidence limits 0.82-0.92) in the training group and 0.79 (0.69-0.88) in the test group. It is concluded that a completely automated method can be used for the interpretation of V-P scintigrams. The performance of this method is similar to others previously presented, whereby features were extracted manually.

A new radiographic method for evaluating the degree of sliding in devices used in hip-fracture surgery.

PURPOSE: To find a practical method for estimating the degree of sliding that occurs in screw-plate devices used in hip-fracture surgery. Greater understanding of the sliding mechanisms in different fracture types should improve surgical technique and reduce the failure rate. METHODS AND RESULTS: In dynamic screw-plate devices, the lag screw slides inside the barrel of the plate. A recent innovation allows the barrel-plate to slide inside a side-plate, thus making possible a combined fracture compression along the neck and the shaft of femur. The lengths of the different parts and the angle of a device in vivo, measured on a radiograph, depend on the position of the femur relative to the photographic film and the roentgen source. We obtained these measurements with a ruler and a protractor from sequential a.p. radiographs of the hip and implemented them in a special computerized program that used the principles of the scaled orthographic and the central projection models. These calculations provided the correct amount of sliding by the lag screw and by the barrel-plate within the side-plate. CONCLUSION: The method presented here can establish the real degree of sliding in screw-plate devices from standard a.p. radiographs independently of the position of the hip.

Lack of prognostic significance for p53-overexpression and Ki-67-immunoreactivity in oral T1-2 squamous cell carcinomas.

Previous investigations on squamous cell carcinomas (SCC) of the head and neck region have failed to reveal a significant correlation between p53-overexpression or Ki-67-immunoreactivity and survival. Contrary to these studies we restricted the evaluation to T1-2 SCC from the oral cavity. Immunohistochemically identified p53-overexpression was observed in 69% of the tumours, and Ki-67-positive cancer cells ranged from 12 to 83% in individual rumours (median 37%). No significant correlation was found between p53-overexpression or Ki-67-positivity and survival. Although the degree of tumour differentiation and the pattern of invasion correlated with prognosis (p=0.0387 and 0.0319 respectively), these associations were too weak to be used as prognostic markers.

Detection of human papillomavirus in routinely processed biopsy specimens from laryngeal papillomas: evaluation of reproducibility of polymerase chain reaction and DNA in situ hybridization procedures.

The frequency of human papillomavirus (HPV) in laryngeal papillomas varies largely among different studies. DNA in situ hybridization (ISH) has been the most widely used method for detection of HPV. The aim of this study was to compare the reproducibility and sensitivity of ISH with polymerase chain reaction (PCR) in 35 specimens of laryngeal papillomas routinely fixed in buffered or unbuffered formalin. Out of 12 specimens fixed in buffered formalin, 10 were positive for HPV 6/11 using ISH. The procedure was repeated three times and three specimens were positive only twice. Nine biopsies were positive for HPV using PCR with consensus primers (My 09/11) on dewaxed tissue without extracting DNA. In three repeated PCRs, the results were inconsistent in three samples. After DNA extraction, all 12 samples were positive with PCR. Of the 23 specimens fixed in unbuffered formalin, 14 were HPV-positive with ISH, while only one was positive with PCR. We concluded that PCR with My 09/11 consensus primers is a highly sensitive method for detection of HPV in laryngeal papillomas fixed in buffered formalin, but useless for samples fixed in unbuffered formalin. When DNA was extracted from the former type of fixed tissue, the results were highly reproducible. In contrast to PCR, ISH appeared to be less influenced by fixation procedure, but it was not as reproducible and sensitive as PCR. Negative results did not necessarily mean absence of HPV.

Transphrenic dissemination of actinomycosis.

Thoracic actinomycosis is an uncommon disease and often presents difficulty in diagnosis. Two cases are presented in which thoracic actinomycosis produced fistulae between the thoracic and abdominal cavities. Surgical drainage and high dose penicillin for at least 4-6 months was the treatment of choice.

Going Dutch in nocturnal enuresis.

Based on several intervention programmes, a strategy for the treatment of nocturnal enuresis has recently been developed by an expert committee in the Netherlands. It consists of three parts. First, two structured interviews are given: one to differentiate between enuresis and incontinence and one to detect associated problems such as diurnal enuresis, constipation or behavioural problems. Secondly, a medical examination is made, confined to the inspection of the external genitalia and lower back, palpation of the abdomen and urine examination. Thirdly, the following guidelines for treatment at different age levels are applied: up to the age of 6 years no intervention is needed; between the ages of 6 and 8 years, lifting out of bed and/or the calendar method; between the ages of 8 and 12 years, enuresis alarm (if not successful, medication with desmopressin is prescribed for a restricted period of time), and ambulatory dry-bed training in a group setting may follow; over 13 years of age, clinical dry-bed training according to the Messer/Azrin method is advised. According to the expert committee, these guidelines offer sufficient possibilities to deal with the problem of nocturnal enuresis.

Topical application of 12-O-tetradecanoylphorbol-13-acetate induces dyssynchronous expression of keratins K1 and K10 in mouse epidermis.

12-O-tetradecanoylphorbol-13-acetate (TPA) is a potent tumor promoter that causes severe alterations in the biosynthesis of epidermal keratins. This study shows that TPA induces a dyssynchronous effect on keratin expression in stratified squamous epithelium. The effect of TPA on the separate expression of the maturation-associated keratins K1 and K10 was studied by immunohistochemistry in an unperturbed replicative keratinocyte population of hairless mice epidermis in relation to changes in the cell cycle time during regeneration. Keratinocytes in DNA synthesis were pulse-labeled by intraperitoneal injection of the thymidine analogue 5-bromodeoxyuridine (BrdUrd) 1 h before a single topical application of TPA. The BrdUrd-labeled cell cohort, representing an originally unperturbed replicative keratinocyte population exposed to TPA mainly in the postreplicative period, was followed for up to 97 h. The results suggested unaltered timing of the onset of K1 and K10 expression compared with normal epidermis (18 and 24 h, respectively, following DNA synthesis). This indicates that the synthesis of both keratins was programmed before the keratinocytes entered their last DNA synthesis. A reduction in K10 expression from about 30 h compared with that of K1 expression was observed. Mathematical modeling suggested a delay in K10 expression related to the second and third rounds of cell divisions after pulse-labeling. How TPA induces such dyssynchrony in K1 and K10 regulation remains unknown.

Topical application of calcitriol alters expression of filaggrin but not keratin K1 in mouse epidermis.

Calcitriol (1 alpha,25-dihydroxyvitamin D3) and its analogues are antiproliferative agents which promote epidermal differentiation in vitro, possibly reflecting their modes of action in the treatment of psoriasis. We examined the effect of calcitriol on early and late terminal differentiation in mouse epidermis in vivo using an immunofluorescence assay to detect keratin K1 and filaggrin expression. Pulse labelling with the tymidine analogue 5-bromo-2-deoxyuridine (BrdUrd) was performed by intraperitoneal injection of mice immediately or 16 h after a single topical application of 0.72 nmol calcitriol. The BrdUrd labelling index (LI) and keratin K1 or filaggrin expression of postmitotic cell cohorts were scored by paired immunofluorescence staining for up to 72 h after BrdUrd labelling. Calcitriol induced cell proliferation as shown by a 100% increase in the BrdUrd LI 17 h after application. The onset of keratin K1 expression in the postmitotic period was, however, unchanged in both series after calcitriol treatment. Filaggrin expression appeared earlier after calcitriol treatment than in control epidermis, probably reflecting altered cell kinetics with increased epidermal turnover. The results suggest that calcitriol only influences the later stages of the keratinocyte differentiation programme, possibly secondarily to its hyperproliferative effect.

Gene activation studied by immunological methods.

Gene activation can be studied at several levels: transcription (mRNA), translation (proteins), or phenotypical alterations (functional activity or morphology). These levels can be studied in situ or biochemically by the use of specific probes for normal or altered DNA, mRNA, or proteins. Immunological probes are potent tools for studies of alterations induced by xenobiotics in target organs. When the effects of xenobiotics are studied in whole tissue, the cellular heterogeneity of the organ must be taken into account. For this reason, combined in situ and biochemical techniques are necessary. Antibodies to normal or altered cellular constituents are used for identification, quantitation, and cellular localization of proteins and modified DNA. Many xenobiotics alter gene activation by interactions with DNA. After activation, 2-acetylaminofluorene (AAF) forms DNA adducts, which can be identified immunologically. Combined with bromodeoxyuridine (BrdU) pulse labeling, techniques have been developed to demonstrate reduced adduct concentrations in proliferating cells and preneoplastic foci in the livers of AAF-fed rats. Carcinogen-induced DNA modifications are implicated as a major mechanism of altered gene activation in neoplasia, leading to phenotypical alterations. Also, cellular differentiation may be affected by xenobiotics. Differentiation-associated markers can be used for studies of gene activation. In mouse skin, the keratins K1 and K10 are only expressed in suprabasal, differentiating cells. BrdU pulse chase experiments combined with double immunofluorescence have revealed that K1 and K10 are sequentially turned on 18 to 24 hr after DNA synthesis and are followed by suprabasal migration. After a single application of the tumor promotor 12-O-tetradecanoylphorbol-13-acetate (TPA), cell migration starts directly after mitosis.(ABSTRACT TRUNCATED AT 250 WORDS)

Differential effects of topical retinoic acid application on keratin K1 and filaggrin expression in mouse epidermis.

Retinoic acid (RA) modulates epidermal homeostasis and affects differentiation-associated proteins such as keratin K1 and filaggrin. Because results from in vitro and in vivo studies have been conflicting with respect to RA effects on keratinization, we examined the terminal differentiation of epidermal cell cohorts after RA stimulation in vivo. Pulse-labelling with 5-bromo-2-deoxyuridine (BrdU) was performed by intraperitoneal injection of mice immediately or at 16 h after a single topical application of 100 nmol RA. The cell cohort labelled at the time of RA application consisted of previously unperturbed cells exposed to RA after initiation of S-phase, whereas the cohort labelled 16 h after RA application consisted of cells stimulated into the S-phase by RA. These two cohorts of partially synchronized cells were followed for up to 72 h after BrdU labelling. Such labelling combined with keratin K1 or filaggrin expression was scored by paired immunofluorescence staining of skin sections. The onset of keratin K1 expression was unchanged in both series after RA treatment, while filaggrin appeared earlier than in controls. The differential effect of RA on the maturation markers was related to the proliferative activity, the increased cell turnover, and the shortened epidermal transit time. The onset of keratin expression appeared to be regulated before the postmitotic period, whereas filaggrin expression appeared to be regulated during the late phase of the maturation process, thus being influenced by the actual epidermal kinetics and structural alterations. These results suggested that the effect of RA on epidermal differentiation is secondary to its effect on proliferation, as determined by the altered cellular age distribution following regenerative proliferation.

Application of cantharidin or 12-O-tetradecanoylphorbol-13-acetate on mouse epidermis induces a cell population shift that causes altered keratin distribution.

The tumour promoter 12-O-tetradecanoyl-phorbol-13-acetate (TPA) causes changes in epidermal protein expression, especially in the major differentiation products keratins K1 and K10. These keratins and filaggrin were studied in a pulse-labelled cell cohort in hairless mouse epidermis stimulated to proliferate by TPA or the hyperplasiogen cantharidin. Cells in DNA synthesis were pulse-labelled by 5-bromo-2-deoxyuridine (BrdU) 16 h after topical application of cantharidin or TPA. The BrdU-labelled cell cohort, the two keratins, and filaggrin were spatially mapped by paired immunofluorescence staining. Both cantharidin- and TPA-treated epidermis displayed altered distributions of K1 and K10 with expression only in the outermost cell layers, but the start of their postreplicative expression paralleled that in normal epidermis (18 h for K1 and 24 h for K10 after the last round of DNA synthesis). Cantharidin- and TPA-induced epidermal hyperplasia showed increased basal cell proliferation, accelerated suprabasal migration, and shortened transit time. Thus, the newly formed hyperplastic epidermis was composed of keratinocytes with a lower mean cellular age than that seen in unperturbed epidermis, which caused altered distribution of K1 and K10. Both hyperplastic and normal epidermis showed filaggrin expression in stratum granulosum; this started earlier in treated (30-36 h) than in untreated (96 h) skin. We concluded that the postmitotic onset of K1 and K10 expression was unaltered in regenerative epidermis, whereas filaggrin expression was considerably accelerated and thus influenced by the cell kinetic changes.

Expression of keratins K6 and K16 in regenerating mouse epidermis is less restricted by cell replication than the expression of K1 and K10.

Biosynthesis of the hyperproliferation-associated keratins K6 and K16 was studied in mouse epidermis following a single topical application of the tumour promoter 12-O-tetradecanoylphorbol-13-acetate (TPA). An epidermal cell cohort was followed by pulse-labelling with the thymidine analogue 5-bromodeoxyuridine (BrdUrd). Mice were injected intraperitoneally with BrdUrd 1 h before a single topical application of TPA. TPA induced regenerative epidermal hyperplasia with hyperproliferation and shortened suprabasal transit time. The BrdUrd-labelled cell cohort was followed for 96 h after TPA-treatment by two-colour immunofluorescence staining with antibodies to BrdUrd, keratin K6 and K16. In control animals, K6 and K16 were found only in the hair follicles. K6 expression was immediately induced in all epidermal cell layers of TPA-treated epidermis, including actively replicating cells and it was expressed during the whole observation period. K16 was only present in post-mitotic cells and was transiently expressed 8-72 h after TPA treatment. Our results suggest that the expression of K6 and K16 is less restricted by cellular replication than the normally occurring K1 and K10 keratins.

Vigabatrin in the treatment of infantile spasms.

The anti-epileptic effect of vigabatrin as a first choice therapy was investigated in 6 children suffering from infantile spasms (IS). All 6 children showed a reduction in seizure frequency. Three children became seizure-free within a period of 2 weeks. Because our results suggest "an all or nothing phenomenon", a period of 2 weeks may be sufficient to evaluate the efficacy of vigabatrin in untreated infants suffering from IS. We suggest to use vigabatrin as a first choice anti-epileptic drug in infants with IS.