Isolation, characterization and binding properties of two rat liver fatty acid-binding protein isoforms

Mammalian liver has only one fatty acid-binding protein (L-FABP) while the liver of non-mammalian vertebrates expresses a liver basic FABP (Lb-FABP) in addition to other members of the FABP family. We explore the possibility that L-FABP isoforms accomplish, in the liver of mammals, the metabolic functions corresponding to the different FABPs present in the liver of non-mammalian vertebrates. We have isolated isoforms I and II which have a different residue 105, Asn in the former and Asp in the latter. We made a conformational comparison of the apo-isoforms by intrinsic fluorescence emission and fourth-derivative spectroscopy, native-state proteolysis and unfolding curves. Ligand affinity was studied by measuring cis-parinaric acid displacement by different ligands. They have differences in their molecular conformation, including the environment of the binding site. Isoform II has probably a more open conformation than isoform I, thus allowing the binding of a greater variety of ligands. The affinity of isoform II for lysophospholipids, prostaglandins, retinoids, bilirubin and bile salts is greater than that of isoform I. These characteristics of rat L-FABP isoforms I and II suggest that they may accomplish different functions as happens with those of the different FABP types in non-mammalian species.     

Biogenesis and function of the lipidic structures of pollen grains

 Pollen grains contain several lipidic structures, which play a key role in their development as male gametophytes. The elaborate extracellular pollen wall, the exine, is largely formed from acyl lipid and phenylpropanoid precursors, which together form the exceptionally stable biopolymer sporopollenin. An additional extracellular lipidic matrix, the pollen coat, which is particularly prominent in entomophilous plants, covers the interstices of the exine and has many important functions in pollen dispersal and pollen-stigma recognition. The sporopollenin and pollen coat precursors are both synthesised in the tapetum under the control of the sporophytic genome, but at different stages of development. Pollen grains also contain two major intracellular lipidic structures, namely storage oil bodies and an extensive membrane network. These intracellular lipids are synthesised in the vegetative cell of the pollen grain under the control of the gametophytic genome. Over the past few years there has been significant progress in elucidating the composition, biogenesis and function of these important pollen structures. The purpose of this review is to describe these recent advances within the historical context of research into pollen development. Received: 1 November 1997 / Revision accepted: 3 February 1998     

Interleukin-5 modulates interleukin-8 secretion in eosinophilic inflammation.

Serum and BALF (bronchoalveolar lavage fluid) IL-8 levels and serum levels were investigated in Toxocara canis infected guinea-pigs and the role of IL-5 as a modulator of cytokine secretion was studied. Serum levels increased early in infected animals, exceeding control levels 4 h after infection, peaked between days 6 and 18, and continued to exceed control levels after 48 days of infection. Serum and BALF IL-8 levels showed the same profile as blood eosinophilia, increasing 6 days post-infection and peaking between days 18 and 24. Treatment of infected animals with anti-IL-5 Ab suppressed eosinophilia with a parallel increase in blood IL-8 levels, whereas no change was found in levels. To support our in vivo observation we carried out experiments in vitro using guinea-pig LPS-stimulated adherent peritoneal cells which release large amounts of IL-8 into the supernatants. When rIL-5 was added to LPS-stimulated cells, 65% inhibition of IL-8 release into the supernatants was observed. Pre-incubation of cells with anti-IL-5 Ab prevented the inhibition of IL-8 release into the supernatants induced by rIL-5. Our results demonstrate for the first time that TNF-alpha and IL-8 are released concomitant with or after IL-5 in the eosinophilic inflammation induced by T. canis. Moreover, in addition to showing that IL-5 is fundamental for the induction of blood eosinophilia, the present results suggest that this cytokine may play a new biological role by acting as modulator of IL-8 secretion.     

Cerebral Circulation Time is Prolonged and Not Correlated with EDSS in Multiple Sclerosis Patients: A Study Using Digital Subtracted Angiography

Literature has suggested that changes in brain flow circulation occur in patients with multiple sclerosis. In this study, digital subtraction angiography (DSA) was used to measure the absolute CCT value in MS patients and to correlate its value to age at disease onset and duration, and to expand disability status scale (EDSS). DSA assessment was performed on eighty MS patients and on a control group of forty-four age-matched patients. CCT in MS and control groups was calculated by analyzing the angiographic images. Lesion and brain volumes were calculated in a representative group of MS patients. Statistical correlations among CCT and disease duration, age at disease onset, lesion load, brain volumes and EDSS were considered. A significant difference between CCT in MS patients (mean = 4.9s; sd = 1.27s) and control group (mean = 2.8s; sd = 0.51s) was demonstrated. No significant statistical correlation was found between CCT and the other parameters in all MS patients. Significantly increased CCT value in MS patients suggests the presence of microvascular dysfunctions, which do not depend on clinical and MRI findings. Hemodynamic changes may not be exclusively the result of a late chronic inflammatory process.     

Enhanced Amphiphilic Profile of a Short β-Stranded Peptide Improves Its Antimicrobial Activity

SB056 is a novel semi-synthetic antimicrobial peptide with a dimeric dendrimer scaffold. Active against both Gram-negative and -positive bacteria, its mechanism has been attributed to a disruption of bacterial membranes. The branched peptide was shown to assume a β-stranded conformation in a lipidic environment. Here, we report on a rational modification of the original, empirically derived linear peptide sequence [WKKIRVRLSA-NH₂, SB056-lin]. We interchanged the first two residues [KWKIRVRLSA-NH₂, β-SB056-lin] to enhance the amphipathic profile, in the hope that a more regular β-strand would lead to a better antimicrobial performance. MIC values confirmed that an enhanced amphiphilic profile indeed significantly increases activity against both Gram-positive and -negative strains. The membrane binding affinity of both peptides, measured by tryptophan fluorescence, increased with an increasing ratio of negatively charged/zwitterionic lipids. Remarkably, β-SB056-lin showed considerable binding even to purely zwitterionic membranes, unlike the original sequence, indicating that besides electrostatic attraction also the amphipathicity of the peptide structure plays a fundamental role in binding, by stabilizing the bound state. Synchrotron radiation circular dichroism and solid-state ¹⁹F-NMR were used to characterize and compare the conformation and mobility of the membrane bound peptides. Both SB056-lin and β-SB056-lin adopt a β-stranded conformation upon binding POPC vesicles, but the former maintains an intrinsic structural disorder that also affects its aggregation tendency. Upon introducing some anionic POPG into the POPC matrix, the sequence-optimized β-SB056-lin forms well-ordered β-strands once electro-neutrality is approached, and it aggregates into more extended β-sheets as the concentration of anionic lipids in the bilayer is raised. The enhanced antimicrobial activity of the analogue correlates with the formation of these extended β-sheets, which also leads to a dramatic alteration of membrane integrity as shown by ³¹P-NMR. These findings are generally relevant for the design and optimization of other membrane-active antimicrobial peptides that can fold into amphipathic β-strands.     

Impaired Increase of Plasma Abscisic Acid in Response to Oral Glucose Load in Type 2 Diabetes and in Gestational Diabetes

The plant hormone abscisic acid (ABA) is present and active in humans, regulating glucose homeostasis. In normal glucose tolerant (NGT) human subjects, plasma ABA (ABAp) increases 5-fold after an oral glucose load. The aim of this study was to assess the effect of an oral glucose load on ABAp in type 2 diabetes (T2D) subjects. We chose two sub-groups of patients who underwent an oral glucose load for diagnostic purposes: i) 9 treatment-naive T2D subjects, and ii) 9 pregnant women with gestational diabetes (GDM), who underwent the glucose load before and 8–12 weeks after childbirth. Each group was compared with matched NGT controls. The increase of ABAp in response to glucose was found to be abrogated in T2D patients compared to NGT controls. A similar result was observed in the women with GDM compared to pregnant NGT controls; 8–12 weeks after childbirth, however, fasting ABAp and ABAp response to glucose were restored to normal in the GDM subjects, along with glucose tolerance. We also retrospectively compared fasting ABAp before and after bilio-pancreatic diversion (BPD) in obese, but not diabetic subjects, and in obese T2D patients, in which BPD resulted in the resolution of diabetes. Compared to pre-BPD values, basal ABAp significantly increased 1 month after BPD in T2D as well as in NGT subjects, in parallel with a reduction of fasting plasma glucose. These results indicate an impaired hyperglycemia-induced ABAp increase in T2D and in GDM and suggest a beneficial effect of elevated ABAp on glycemic control.     

Treatment of Solitary Painful Osseous Metastases with Radiotherapy,Cryoablation or Combined Therapy: Propensity Matching Analysis in 175 Patients

Purpose

aim of this study was to identify outcomes in pain relief and quality of life in patients with a solitary painful osseous metastasis treated by radiotherapy, cryoablation or the combination using a propensity score matching study design.

Materials and Methods

175 patients with painful bone metastases were included in the study. Twenty-five of them underwent a radiation course (20 Gy in five daily fractions) 15 days after the cryoablation. These subjects were retrospectively matched by propensity analysis with a group of subjects treated by radiotherapy (125 subjects) and with a group treated byCryoablation (25 subjects). The pain relief in terms of complete response, rate of subjects requiring analgesics after treatments and the changes in self-rated quality of life were measured. Informed consent was obtained from the subject and the study was approved by the local Ethical Committee.

Results

An higher proportion of subjects treated by cryoablation (32%) or cryoablation followed by RT (72%;) experienced a complete response compared with patients treated by radiotherapy alone (11.2%). After Bonferroni correction strategy, the addition of radiotherapy to cryoablation significantly improved the rate of complete response compared with cryoablation alone (p = 0.011) and this paralleled with an improved self-rated quality of life. Seventeen subjects (13.6%) of patients in the radiotherapy group, 9 (36%) in the cryoablation group, and 19 (76)% in the cryoablation- radiotherapy group did not require narcotic medications.

Conclusions

The addition of radiotherapy to cryoablation favorably impacts on perceived pain, with a favorable toxicity profile. However, our data should be interpreted with caution and could serve as a framework around which to design future trials.     

Multi-Target Analysis and Design of Mitochondrial Metabolism

Analyzing and optimizing biological models is often identified as a research priority in biomedical engineering. An important feature of a model should be the ability to find the best condition in which an organism has to be grown in order to reach specific optimal output values chosen by the researcher. In this work, we take into account a mitochondrial model analyzed with flux-balance analysis. The optimal design and assessment of these models is achieved through single- and/or multi-objective optimization techniques driven by epsilon-dominance and identifiability analysis. Our optimization algorithm searches for the values of the flux rates that optimize multiple cellular functions simultaneously. The optimization of the fluxes of the metabolic network includes not only input fluxes, but also internal fluxes. A faster convergence process with robust candidate solutions is permitted by a relaxed Pareto dominance, regulating the granularity of the approximation of the desired Pareto front. We find that the maximum ATP production is linked to a total consumption of NADH, and reaching the maximum amount of NADH leads to an increasing request of NADH from the external environment. Furthermore, the identifiability analysis characterizes the type and the stage of three monogenic diseases. Finally, we propose a new methodology to extend any constraint-based model using protein abundances.