Root antioxidant responses of two Pisum sativum cultivars to direct and induced Fe deficiency.

The contribution of antioxidant defence systems in different tolerance to direct and bicarbonate-induced Fe deficiency was evaluated in two pea cultivars (Kelvedon, tolerant and Lincoln, susceptible). Fe deficiency enhanced lipid peroxidation and H2 O2 concentration in roots of both cultivars, particularly in the sensitive one grown under bicarbonate supply. The results obtained on antioxidant activities (SOD, CAT, POD) suggest that H2 O2 accumulation could be due to an overproduction of this ROS and, at the same time, to a poor capacity to detoxify it. Moreover, under bicarbonate supply the activity of POD isoforms was reduced only in the sensitive cultivar, while in the tolerant one a new isoform was detected, suggesting that POD activity might be an important contributor to pea tolerance to Fe deficiency. The presence of bicarbonate also resulted in stimulation of GR, MDHAR and DHAR activities, part of the ASC-GSH pathway, which was higher in the tolerant cultivar than in the sensitive one. Overall, while in the absence of Fe only slight differences were reported between the two cultivars, the adaptation of Kelvedon to the presence of bicarbonate seems to be related to its greater ability to enhance the antioxidant response at the root level.

A green and bio-inspired process to afford durable anti-biofilm properties to stainless steel.

A bio-inspired durable anti-biofilm coating was developed for industrial stainless steel (SS) surfaces. Two polymers inspired from the adhesive and cross-linking properties of mussels were designed and assembled from aqueous solutions onto SS surfaces to afford durable coatings. Trypsin, a commercially available broad spectrum serine protease, was grafted as the final active layer of the coating. Its proteolytic activity after long immersion periods was demonstrated against several substrata, viz. a synthetic molecule, N-α-benzoyl-DL-arginine-p-nitroanilide hydrochloride (BAPNA), a protein, FTC-casein, and Gram-positive biofilm forming bacterium Staphylococcus epidermidis.

Biomolecule-based antibacterial coating on a stainless steel surface: multilayer film build-up optimization and stability study.

The goal of this paper was to establish the durability profile of antibacterial multilayer thin films under storage and usage conditions. Thin films were built on stainless steel (SS) by means of a layer-by-layer process alternating a negatively charged polyelectrolyte, polyacrylic acid, with a cationic antibacterial peptide, nisin. SS coupons coated with the antibacterial film were challenged under environmental and usage conditions likely to be encountered in real-world applications. The change in antibacterial activity elicited by the challenge was used as an indicator of multilayer film resistance. Antibacterial SS samples could be stored for several weeks at 4°C in ambient air and antibacterial films were resistant to dipping and mild wiping in water and neutral detergent. The multilayer coating showed some weaknesses, however, that need to be addressed.

Biomolecules in multilayer film for antimicrobial and easy-cleaning stainless steel surface applications.

Microorganisms are able to attach to, grow on, and ultimately form biofilms on a large variety of surfaces, such as industrial equipment, food contact surfaces, medical implants, prostheses and operating rooms. Once organized into biofilms, bacteria are difficult to remove and kill, which increases the risk of cross-contamination and infection. One way to address the problem may thus be to develop antibacterial, anti-adhesion, 'easy cleaning' surfaces. In this study, stainless steel (SS) surfaces with antibacterial properties were created by embedding several antimicrobial peptides in a multilayer film architecture. The biocidal effect of these surfaces was demonstrated against both Gram-positive and Gram-negative bacteria according to two ISO tests. Also, coating SS surfaces with either mucin or heparin led to a reduction of S. epidermidis adhesion of almost 95% vs the bare substratum. Finally, by combining both antibacterial and anti-adhesion biomolecules in the same multilayer film, SS surfaces with better cleanability were produced. This surface coating property may help to delay the buildup of a dead bacterial layer which is known to progressively reduce exposure of the coating, leading to an undesirable decrease in the antibacterial effect of the surface.

An abnormal mRNA produced by a novel PMP22 splice site mutation associated with HNPP.

Hereditary neuropathy with liability to pressure palsies (HNPP) is an autosomal dominant, demyelinating neuropathy. Point mutations in the PMP22 gene are a rare cause of HNPP. A novel PMP22 splice site mutation (c.179+1 G-->C) is reported in an HNPP family. By reverse transcriptase-polymerase chain reaction experiments, this mutation was shown to cause the synthesis of an abnormal mRNA in which a premature stop codon probably produces a truncated non-functional protein.

[Behçet's disease in children: cortical calcifications]. / Enfermedad de Behçet en la infancia: calcificaciones corticoencefálicas.

INTRODUCTION: Between 5 and 48% of the patients with Behçet s disease have neurological abnormalities. In children, meningoencephalitis as an initial manifestation of the disease is not frequent. CASE REPORT: We present a four years old girl with a Behçet s disease. When she was six months of age, during an hospitalization caused by malnutrition and respiratory infection, she had developed an aseptic meningoencephalitis and laboratory test showed hypergammaglobulinemia. When she was 21 months old, she had seizures as epilepticus status, and good response was achieve treating with anticonvulsive drugs. She did not repeat episodes of epileptic seizures, but the patient showed language and neuromotor problems. CT was abnormal with asymmetric ventricular dilatation and increased subarachnoides cortical sulci with multiple calcifications in those areas. She also suffered respiratory and gastrointestinal infections that required hospitalization. At the age of 3 years and 6 months, she had occular affection with uveitis, oral and genital ulceration. The clinical history of the girl consisting of meningoencephalitis, gastrointestinal and respiratory disorders, later presentation of uveitis and oral and genital ulcers confirmed the Behçet s disease diagnosis. CONCLUSION: We present a girl with multisystemic disorders characteristic of the Behçet s disease. Initially she had an aseptic meningoencephalitis developing cortical calcifications.

Weekly gemcitabine plus Epirubicin as effective chemotherapy for advanced pancreatic cancer: a multicenter phase II study.

The current role of chemotherapy in pancreatic carcinoma is limited, and progress in the treatment of this disease represents a significant challenge to medical oncology. The most promising drug under study is gemcitabine, a relatively new antimetabolite that represents an attractive candidate for combination chemotherapy because of its excellent side-effect profile and the absence of overlapping toxicities with other chemotherapeutic agents. Combined administration of gemcitabine and anthracyclines could result in the induction of DNA breaks that are not easily repaired by the cell's machinery, thus enhancing the apoptotic signals triggered by these lesions. Forty-four patients with locally advanced and/or metastatic pancreatic adenocarcinoma were enrolled in this multicenter study. Patients received Epirubicin 20 mg m(-2) for 3 weeks followed by 1 week of rest (1 cycle) and gemcitabine 1000 mg m(-2) after Epirubicin on the same day. All were assessable for toxicity and response, 11 patients responded to treatment with one complete response and 10 partial responses, for an overall response rate of 25%. Median survival was 10.9 months (range, 2-26 months). Therapy was well tolerated, with a low incidence of haematologic grade >2 toxicity. A total of 12 of 27 (44.4%) eligible patients attained a clinical benefit response. Our findings suggest that the gemcitabine-epirubicin schedule is active and well tolerated in patients with advanced pancreatic cancer.

Enfermedad de Behçet en la infancia:calcificaciones corticoencefálicas / Behçet's disease in children: cortical calcifications

Introducción. Entre el 5 y el 48 por ciento de los pacientes con enfermedad de Behçet, presentan alteraciones neurológicas. La meningoencefalitis en la lactancia es infrecuente como manifestación inicial. Caso clínico. Presentamos una niña de 4 años y 10 meses de edad, que a los 6 meses de vida padecía meningoencefalitis aséptica, después de una prolongada internación por un cuadro de desnutrición moderada y neumopatía. Se constató hipergammaglobulinemia. Al año y 9 meses de vida presentó status convulsivo. Posteriormente las crisis epilépticas se controlaron. La paciente presenta un retraso neuromotor y lingüístico. La tomografía axial computarizada demuestra una dilatación ventricular asimétrica, con un aumento de los espacios subaracnoideos corticales que presentaban calcificaciones múltiples. Fue internada en numerosas ocasiones por intercurrencias respiratorias y gastrointestinales. A los 3 años y 6 meses de vida presentaba una afección ocular, uveítis, lesiones bucales y genitales. La historia previa de la niña, su cuadro de meningoencefalitis, trastornos gastrointestinales y respiratorios, la aparición de uveítis, úlceras perianales y bucales e hipergammaglobulinemia corresponden a las características clínicas de la enfermedad de Behçet. Conclusión. Se presenta una niña con alteraciones multisistémicas características de la enfermedad de Behçet, afectada de meningoencefalitis precoz con aparición de calcificaciones corticales (AU)

Force measurements on single molecular contacts through evanescent wave microscopy.

We introduce a new method to apply controlled forces on single molecules. The motion of a micron-sized bead attached to a solid surface through a single molecular contact is tracked by evanescent wave microscopy as a force is exerted through a flow. We report measurements of the streptavidin-biotin bond rupture force obtained with this technique. We also obtain detailed measurements of the balance of forces involved in detaching an adhering bead with a flow. A small lateral force translates into a much bigger normal force on the attachment point. This effect is relevant for the interpretation of common cell adhesion assays.

Evaluating ambulatory urodynamics: a prospective study in asymptomatic women.

OBJECTIVE: To identify abnormal detrusor contractions in asymptomatic women undergoing ambulatory urodynamics, to standardise the investigation technique for women with urinary symptoms for clinical practice. DESIGN: Prospective observational study. SETTING: Two tertiary referral urogynaecology units: one in London, UK and one in Varese, Italy. METHODS: Women without urinary symptoms were recruited into the study. Ambulatory urodynamics was performed according to the King's College Hospital protocol. All the traces were analysed in four different ways: 1. without the second bladder transducer measurements displayed on the screen and without the diary; 2. as in 1. but with the diary; 3. with the second bladder sensor displayed on the screen but without the diary; 4. as in 3. but with the diary. The diary was always interpreted at the end of the test with the woman present. For each method we reported the total number of detrusor contractions and whether the woman would have been diagnosed as having abnormal detrusor contraction. Cochran's Q test was used for statistical analysis. RESULTS: Twenty-six women (mean age 31.7 years) were recruited. Ambulatory urodynamic testing lasted an average of 3.57 hour. The diagnosis of abnormal detrusor contraction varied by 65.4% depending on the method of analysis used. The diary and the women's symptoms during the test were the most discriminating factors. A significant difference between the diagnoses was obtained when comparing the four different ways of analysis. According to our protocol only three women (11.5%) had abnormal detrusor contractions. CONCLUSIONS: Using our protocol almost 90% of asymptomatic women have a stable bladder on ambulatory urodynamics. This is similar to the results of labouratory urodynamic testing of asymptomatic women but is in contrast to previous reports of ambulatory urodynamics of asymptomatic women. The technique used during ambulatory urodynamics determines the tests ability to detect detrusor instability with an accurate symptom diary being the most important.

Phosphoenolpyruvate carboxylase in cucumber (Cucumis sativus L.) roots under iron deficiency: activity and kinetic characterization.

Phosphoenolpyruvate carboxylase (PEPCase) activity was investigated in cucumber roots grown under iron starvation. The enzyme extracted from plants grown in the presence and in the absence of Fe was characterized both kinetically and biochemically. Extractable PEPCase activity was increased by 4-fold in the absence of Fe. This increase began about 5 d after Fe starvation. Western blot analysis revealed the presence of two polypeptides with apparent molecular masses of 103 and 108 kDa. At the beginning both the polypeptides were equally present in the control and in the Fe-deficient roots. After 10 d of Fe starvation the increase was clearly evident and concerned, in particular, the polypeptide of 103 kDa whose enhancement was around 3-fold with respect to the control. Re-supply of iron to Fe-starved roots decreased both the activity and the concentration of the enzyme to the control values. Determination of kinetic parameters revealed that the K:(m) values for the substrates were the same, while the V:(max) was increased by four times for the enzyme extracted from Fe-deficient roots. Also the responses to pH changes and to the allosteric modulators malate and glucose-6-phosphate were different. The kinetic data, the increase in PEPCase specific activity and in the PEPCase polypeptides concentration seem to indicate that under Fe deficiency the enzyme regulation might be, in part, exerted at the transcriptional level.

[Observing an SSU Em 118 dispatch center for continuous quality improvement. The case of SSUEm 118 Varese]. / Una centrale operativa SSU Em 118 a giudizio per verifica e revisione di qualità. Il caso SSU Em 118 Varese.

The present paper highlights quality aspects of the management of an Emergency Health Service Center (SSUEm 118, Varese) in order to identify the corrective measures required in a service that is increasingly close to the citizens real needs and expectations. Data were collected retrospectively on a total 54,301 calls for assistance in the period October 1997-March 1999 from an area covering some 1,300 sq.km with a population of 1,150,000 residents. That resident population was dramatically increased on a daily basis by heavy vehicle traffic particularly on the motorways to the area's many factories and to the Intercontinental Airport Malpensa 2000. The survey employed 7 anaesthetists and resuscitation staff, 14 nurses and 8 Italian Red Cross works from the Emergency Center. The researchers analysed the following phases: call reception and telephone conversation: ambulance dispatch, patient transportation and the alerting of the hospital of destination. The ServFMEA method was used for Quality Control with appropriate dispatch and the conduct and timing of the ambulance service in the Varese SSUEm 118 area. The data collected allowed for a detailed analysis of the accuracy of the information provided over the telephone (over-triage 58%, undertriage 2%), the usefulness of the telephone filter, the colour coding (correct in 40% of cases), pick-up times (5'40" on average) which were related to problems inherent in the ambulance call-out and the way ambulances reached the emergency (BLS 99%, ALS 1%, Air rescue < 1%). It was concluded that Varese SSUEm 118 was effectively and efficiently run in its first 18 months and results were improved as far as they could be given the inadequate funding of the Italian Heatlh Service.

Development of Fe-deficiency responses in cucumber (Cucumis sativus L.) roots: involvement of plasma membrane H(+)-ATPase activity.

One of the mechanisms through which some strategy I plants respond to Fe-deficiency is an enhanced acidification of the rhizosphere due to proton extrusion. It was previously demonstrated that under Fe-deficiency, a strong increase in the H(+)-ATPase activity of plasma membrane (PM) vesicles isolated from cucumber roots occurred. This result was confirmed in the present work and supported by measurement of ATP-dependent proton pumping in inside-out plasma membrane vesicles. There was also an attempt to clarify the regulatory mechanism(s) which lead to the activation of the H(+)-ATPase under Fe-deficiency conditions. Plasma membrane proteins from Fe-deficient roots submitted to immunoblotting using polyclonal antibodies showed an increased level in the 100 kDa polypeptide. When the plasma membrane proteins were treated with trypsin a 90 kDa band appeared. This effect was accompanied by an increase in the enzyme activity, both in the Fe-deficient and in the Fe-sufficient extracts. These results suggest that the increase in the plasma membrane H(+)-ATPase activity seen under Fe-deficiency is due, at least in part, to an increased steady-state level of the 100 kDa polypeptide.

Metabolic responses in cucumber (Cucumis sativus L.) roots under Fe-deficiency: a 31P-nuclear magnetic resonance in-vivo study.

The metabolic responses occurring in cucumber (Cucumis sativus L.) roots (a strategy-I plant) grown under iron-deficiency conditions were studied in-vivo using 31P-nuclear magnetic resonance spectroscopy. Iron starvation induced activation of metabolism leading to the consumption of stored carbohydrates to produce the NAD(P)H, ATP and phosphoenolpyruvate necessary to sustain the increased activity of the NAD(P)H:Fe(3+)-reductase, the H(+)-ATPase (EC 3.6.1.35) and phosphoenolpyruvate carboxylase (EC 4.1.1.31). Activation of catabolic pathways was supported by the enhancement of glycolytic enzymes and concentrations of the metabolites glucose-6-phosphate and fructose-6-phosphate, and by enhancement of the respiration rate. Moreover, Fe-deficiency induced a slight increase in the cytoplasmic (pHc) and vacuolar (pHv) pHs as well as a dramatic decrease in the vacuolar phosphate (Pi) concentration. A comparison was done using fusicoccin (FC), a fungal toxin which stimulates proton extrusion. Changes in pHc and pHv were measured after addition of FC. Under these conditions, a dramatic alkalinization of the pHv of -Fe roots was observed, as well as a concomitant Pi movement from the vacuole to the cytoplasm. These results showed that Fe starvation was indeed accompanied by the activation of metabolic processes useful for sustaining the typical responses occurring at the plasma-membrane level (i.e. increases in the NAD(P)H:Fe(3+)-reductase and H(+)-ATPase activities) as well as those involved in the homeostasis of pHc. The decrease in vacuolar Pi levels induced by Fe-deficiency and FC and movement of Pi from the vacuole to the cytoplasm suggest a possible involvement of this compound in the cellular pH-stat system.

Calcium-dependent phosphorylation regulates the plasma-membrane H(+)-ATPase activity of maize (Zea mays L.) roots.

Phosphorylation/dephosphorylation of the plasma-membrane H(+)-ATPase (EC 3.6.1.35) could act as a regulatory mechanism to control its activity. In this work, a plasmalemma-enriched fraction from maize roots and a partially purified H(+)-ATPase were used to investigate the effects of Ca(2+) and calmodulin on the H(+)-ATPase activity and on its phosphorylation status. Both the hydrolytic and the proton-pumping activities were reduced approximately 50% by micromolar Ca(2+) concentrations while calmodulin did not show any effect either alone or in the presence of Ca(2+). The lack of effect of calmodulin antagonists indicated that calmodulin was not involved in this response. The addition of staurosporine, a kinase inhibitor, abolished the inhibitory effect of Ca(2+). Phosphorylation of plasma membrane and partially purified H(+)-ATPase showed the same behavior. In the presence of Ca(2+) a polypeptide of 100 kDa was phosphorylated. This polypeptide cross-reacted with antibodies raised against the H(+)-ATPase of maize roots. The autoradiogram of the immunodetected protein clearly showed that this polypeptide, which corresponds to the H(+)-ATPase, was phosphorylated. Additional clear evidence comes from the immunoprecipitation experiments: the data obtained show that the H(+)-ATPase activity is indeed influenced by its state of phosphorylation.

Osmotic pressure contribution of albumin to colloidal interactions.

Two surfaces that come in close contact in a solution with macromolecules present experience an attractive force caused by the osmotic pressure. We present a measurement of this effect by using a micrometer-sized sphere bound to a flat plate through a single molecular attachment in an albumin-containing solution. We obtain the osmotic part of the interaction potential with a resolution of <1 nm and a fraction of kTroom. This attractive interaction is seen to have a range comparable to the size of the albumin molecule. The results are broadly in agreement with a geometric model first proposed by Asakura and Oosawa.

Proteins unfold in steps.

I present results from an experiment on the dynamics of folding of a globular protein (bovine serum albumin). Employing a micro-mechanical technique, I perform the measurements on very few molecules (1-100). I observed a sequence of steps in time for both unfolding and refolding. The overall characteristic time of the process is thus built up of waiting times between successive steps. The pattern of steps is reproducible, demonstrating the existence of deterministic pathways for folding and unfolding. Certain symmetries in the patterns of steps may reflect the architecture of the protein's structure.

Metabolic Implications in the Biochemical Responses to Iron Deficiency in Cucumber (Cucumis sativus L.) Roots.

Strategy I plants respond to Fe deficiency by inducing morphological and biochemical modifications at the root level that are apt to make iron available for uptake. Cucumber (Cucumis sativus L.) grown in the absence of Fe has been shown to increase the capacity to acidify the rhizosphere and Fe3+ reduction activity. We have determined in these roots some metabolic activities that might be correlated with the increased proton extrusion. Proton efflux from roots may be followed by a mechanism regulating the cytosolic pH according to the pH-stat theory. Roots grown in the absence of Fe showed an increase in dark 14CO2 fixation and organic acid synthesis and a 6-fold increase in the extractable phosphoenolpyruvate carboxylase activity with respect to the control roots. Dehydrogenase activities producing cytosolic NAD(P)H were also increased under Fe deficiency. The presence of Fe2+, but not Fe3+, inhibited dark 14CO2 fixation in a range between 24 and 52% but did not show any effect on the in vitro phosphoenolpyruvate carboxylase activity.