Homeostatic restitution of cell membranes. Nuclear membrane lipid biogenesis and transport of protein from cytosol to intranuclear spaces

Our studies on homeostatic restitution of cellular and subcellular membranes showed that vesicular intracellular transport is engaged in systematic and coordinated replacement of lipids and proteins in the membranes of the secretory, non-dividing epithelial cells (Slomiany et al., J. Physiol. Pharmacol. 2004; 55: 837-860). In this report, we present evidence on the homeostatic restitution of lipids in the biomembranes that constitute nuclear envelopes. We investigated nuclear membranes lipid synthesis by employing purified intact nuclei (IN), the outer nuclear membrane (ONM), the inner nuclear membrane (INM) and the cell cytosol (CC). In contrast to Endoplasmic Reticulum (ER) which in the presence of CC generates new biomembrane that forms ER vesicles transporting ER products to Golgi, the IN, ONM and INM are not producing transport vesicles. Instead, the newly synthesized lipids remain in the nuclear membranes. The membranes (INM, ONM) of IN incubated with CC become enriched with newly synthesized phosphatidylcholine (PC), phosphatidylinositol (PI), phosphatidylinositol phosphates (PIPs) and phosphatidic acid (PA). The incubation of separated ONM and INM with CC also enriched the membranes with IN specific lipids identified above. Moreover, the incubation of IN or its membranes with CC afforded retention of numerous CC proteins on the nuclear membrane. Here, we concentrated on 30kDa CC protein that displayed affinity to nuclear membrane PIP2. The 30kDa CC protein bound to PIP2 of IN, INM, and ONM. With IN, initially the PIP2-30kDa CC protein complex was detected on ONM, after 30-120 min of incubation, was found on INM and in nuclear contents. At the same time when the 30 kDa protein was released from INM and found in nuclear contents, the PIP2 of INM and ONM became undetectable, while the lipid extract from the membrane displaced from IN contained labeled PI only. Since ONM is an uninterrupted continuum of ER and INM, we speculate that the synthesis of the lipids in the ER, in the region adjacent to nucleus, is defining nuclear outer and inner biomembrane composition, is responsible for transport of the cytosolic protein into the nucleus and, replenishment of ER membrane used for vesicular transport.     

In vivo analgesic and anti-inflammatory activities of ursolic acid and oleanoic acid from Miconia albicans (Melastomataceae)

The aim of this work was to use in vivo models to evaluate the analgesic and anti-inflammatory activities of ursolic acid (UA) and oleanoic acid (OA), the major compounds isolated as an isomeric mixture from the crude methylene chloride extract of Miconia albicans aerial parts in an attempt to clarify if these compounds are responsible for the analgesic properties displayed by this plant. Ursolic acid inhibited abdominal constriction in a dose-dependent manner, and the result obtained at a content of 40 mg kg(-1) was similar to that produced by administration of acetylsalicylic acid at a content of 100 mg kg(-1). Both acids reduced the number of paw licks in the second phase of the formalin test, and both of them displayed a significant anti-inflammatory effect at a content of 40 mg kg(-1). It is noteworthy that the administration of the isolated mixture, containing 65% ursolic acid/35% oleanolic acid, did not display significant analgesic and anti-inflammatory activities. On the basis of the obtained results, considering that the mixture of UA and OA was poorly active, it is suggested that other compounds, rather than UA and OA, should be responsible for the evaluated activities in the crude extract, since the crude extract samples displayed good activities.     

Binding of oxindole-Schiff base copper(II) complexes to DNA and its modulation by the ligand

Previous studies on copper(II) complexes with oxindole-Schiff base ligands have shown their potential antitumor activity towards different cells, inducing apoptosis through a preferential attack to DNA and/or mitochondria. Herein, we better characterize the interactions between some of these copper(II) complexes and DNA. Investigations on its binding ability to DNA were carried out by fluorescence measurements in competitive experiments with ethidium bromide, using plasmidial or calf-thymus DNA. These results indicated an efficient binding process similar to that observed with copper(II)-phenanthroline species, [Cu(o-phen)₂]²⁺, with binding constants in the range 3 to 9 × 10² M^− 1. DNA cleavage experiments in the presence and absence of distamycin, a recognized binder of DNA, indicated that this binding probably occurs at major or minor groove, leading to double-strand DNA cleavage, and being modulated by the imine ligand. Corroborating these data, discrete changes in EPR spectra of the studied complexes were observed in the presence of DNA, while more remarkable changes were observed in the presence of nucleotides (AMP, GMP, CMP or UMP). Additional evidence for preferential coordination of the copper centers to the bases guanine or cytosine was obtained from titrations of these complexes with each nucleotide, monitored by absorption spectral changes. Therefore, the obtained data point out to their action as groove binders to DNA bases, rather than as intercalators or covalent cross-linkers. Further investigations by SDS PAGE using ³²P-ATP or ³²P-oligonucleotides attested that no hydrolysis of phosphate linkage in DNA or RNA occurs, in the presence of such complexes, confirming their main oxidative mechanism of action.     

Interleukin-6 enhances insulin secretion by increasing glucagon-like peptide-1 secretion from L cells and alpha cells

Exercise, obesity and type 2 diabetes are associated with elevated plasma concentrations of interleukin-6 (IL-6). Glucagon-like peptide-1 (GLP-1) is a hormone that induces insulin secretion. Here we show that administration of IL-6 or elevated IL-6 concentrations in response to exercise stimulate GLP-1 secretion from intestinal L cells and pancreatic alpha cells, improving insulin secretion and glycemia. IL-6 increased GLP-1 production from alpha cells through increased proglucagon (which is encoded by GCG) and prohormone convertase 1/3 expression. In models of type 2 diabetes, the beneficial effects of IL-6 were maintained, and IL-6 neutralization resulted in further elevation of glycemia and reduced pancreatic GLP-1. Hence, IL-6 mediates crosstalk between insulin-sensitive tissues, intestinal L cells and pancreatic islets to adapt to changes in insulin demand. This previously unidentified endocrine loop implicates IL-6 in the regulation of insulin secretion and suggests that drugs modulating this loop may be useful in type 2 diabetes.     

People with multiple tattoos and/or piercings are not at increased risk for HBV or HCV in The Netherlands

Background

Although published results are inconsistent, it has been suggested that tattooing and piercing are risk factors for HBV and HCV infections. To examine whether tattooing and piercing do indeed increase the risk of infection, we conducted a study among people with multiple tattoos and/or piercings in the Netherlands who acquired their tattoos and piercings in the Netherlands and/or abroad.

Methods

Tattoo artists, piercers, and people with multiple tattoos and/or piercings were recruited at tattoo conventions, shops (N = 182), and a biannual survey at our STI-outpatient clinic (N = 252) in Amsterdam. Participants were interviewed and tested for anti-HBc and anti-HCV. Determinants of HBV and HCV infections were analysed using logistic regression analysis.

Results

The median number of tattoos and piercings was 5 (IQR 2–10) and 2 (IQR 2–4), respectively. Almost 40% acquired their tattoo of piercing abroad. In total, 18/434 (4.2%, 95%CI: 2.64%–6.46%) participants were anti-HBc positive and 1 was anti-HCV positive (0.2%, 95%CI: 0.01%–1.29%). Being anti-HBc positive was independently associated with older age (OR 1.68, 95%CI: 1.03–2.75 per 10 years older) and being born in an HBV-endemic country (OR 7.39, 95%CI: 2.77–19.7). Tattoo- and/or piercing-related variables, like having a tattoo or piercing in an HBV endemic country, surface percentage tattooed, number of tattoos and piercings etc., were not associated with either HBV or HCV.

Conclusions

We found no evidence for an increased HBV/HCV seroprevalence among persons with multiple tattoos and/or piercings, which might be due to the introduction of hygiene guidelines for tattoo and piercing shops in combination with the low observed prevalence of HBV/HCV in the general population. Tattoos and/or piercings, therefore, should not be considered risk factors for HBV/HCV in the Dutch population. These findings imply the importance of implementation of hygiene guidelines in other countries.     

Prevalence of hepatitis B and C virus infection among leprosy patients in a leprosy-endemic region of central Brazil

Leprosy and hepatitis B virus (HBV) are highly endemic in some regions of the state of Mato Grosso, in central Brazil. The association of leprosy with HBV and hepatitis C virus (HCV) was assessed using a seroprevalence study and 191 leprosy outpatients were included. Demographic data and the clinical classification of leprosy were recorded. Evidence of previous HBV infection was present in 53 patients (27.7%, 95% confidence interval: 21.9-34.5) and two (1%) were HBsAg positive. Five (2.6%) had antibodies to HCV. The prevalence of previous exposure to HBV was higher than expected for an adult population in central Brazil. In contrast, the prevalence of anti-HCV antibodies was not much higher regarding the age range of participants. HBV markers were associated with a higher number of sex partners and the use of injections without proper sterilisation of the syringes. The number of HBV carriers was small, suggesting that there was no increased likelihood of chronification among these patients.     

MIQuant--semi-automation of infarct size assessment in models of cardiac ischemic injury

Background

The cardiac regenerative potential of newly developed therapies is traditionally evaluated in rodent models of surgically induced myocardial ischemia. A generally accepted key parameter for determining the success of the applied therapy is the infarct size. Although regarded as a gold standard method for infarct size estimation in heart ischemia, histological planimetry is time-consuming and highly variable amongst studies. The purpose of this work is to contribute towards the standardization and simplification of infarct size assessment by providing free access to a novel semi-automated software tool. The acronym MIQuant was attributed to this application.

Methodology/Principal Findings

Mice were subject to permanent coronary artery ligation and the size of chronic infarcts was estimated by area and midline-length methods using manual planimetry and with MIQuant. Repeatability and reproducibility of MIQuant scores were verified. The validation showed high correlation (r ^{midline length} = 0.981; r ^area = 0.970 ) and agreement (Bland-Altman analysis), free from bias for midline length and negligible bias of 1.21% to 3.72% for area quantification. Further analysis demonstrated that MIQuant reduced by 4.5-fold the time spent on the analysis and, importantly, MIQuant effectiveness is independent of user proficiency. The results indicate that MIQuant can be regarded as a better alternative to manual measurement.

Conclusions

We conclude that MIQuant is a reliable and an easy-to-use software for infarct size quantification. The widespread use of MIQuant will contribute towards the standardization of infarct size assessment across studies and, therefore, to the systematization of the evaluation of cardiac regenerative potential of emerging therapies.