Nuclear localization of human DNA mismatch repair protein exonuclease 1 (hEXO1)

Human exonuclease 1 (hEXO1) is implicated in DNA mismatch repair (MMR) and mutations in hEXO1 may be associated with hereditary nonpolyposis colorectal cancer (HNPCC). Since the subcellular localization of MMR proteins is essential for proper MMR function, we characterized possible nuclear localization signals (NLSs) in hEXO1. Using fluorescent fusion proteins, we show that the sequence ⁴¹⁸KRPR⁴²¹, which exhibit strong homology to other monopartite NLS sequences, is responsible for correct nuclear localization of hEXO1. This NLS sequence is located in a region that is also required for hEXO1 interaction with hMLH1 and we show that defective nuclear localization of hEXO1 mutant proteins could be rescued by hMLH1 or hMSH2. Both hEXO1 and hMLH1 form complexes with the nuclear import factors importin β/α1,3,7 whereas hMSH2 specifically recognizes importin β/α3. Taken together, we infer that hEXO1, hMLH1 and hMSH2 form complexes and are imported to the nucleus together, and that redundant NLS import signals in the proteins may safeguard nuclear import and thereby MMR activity.     

Civil war and the non-linearity of time: approaching a Mozambican politics of irreconciliation

At least 1 million people died during the Mozambican civil war (1976/7-92). Unfolding after gaining independence from Portugal (1975) and alongside experiments with Afro-socialism in the 1980s, the war, despite its brutality, has not been subjected to global templates of reconciliation processes. Thus it comprises a unique case to probe what irreconciliation might mean – both as a political horizon and as an analytical concept. This text juxtaposes ethnographic material from rural, central Mozambique from the late 1990s and early 2000s emphasizing reconciliation with material from the same spaces from the 2010s onwards, where I identify what I term a ‘politics of irreconciliation’. I will make three arguments. First, informed by Hannah Arendt, I approach irreconciliation as fundamentally about the rejection of a world of violence in search of a world shared in common. Second, drawing on recent anthropological theorizing about temporal regimes and chronopolitics, I argue for the salience of a non-linear understanding of the politics of irreconciliation to grapple with the fact that civil war violence is understood as dangerously uncontained rather than nominally past. Third, within the context of Mozambique, forgiveness and its other, irreconciliation, are not only intimately tied to the temporally past or present; they are also, as I show, produced by a tangible and intense absence of a productive future.     

Wound Healing Properties of Commercial Milk Hydrolysates in Intestinal Cells

International Journal of Peptide Research and Therapeutics - Eight commercial milk protein preparations of whey protein or casein enzymatic hydrolysates were investigated for migration and...     

The SNARE protein vti1a functions in dense‐core vesicle biogenesis

The SNARE protein vti1a is proposed to drive fusion of intracellular organelles, but recent data also implicated vti1a in exocytosis. Here we show that vti1a is absent from mature secretory vesicles in adrenal chromaffin cells, but localizes to a compartment near the trans‐Golgi network, partially overlapping with syntaxin‐6. Exocytosis is impaired in vti1a null cells, partly due to fewer Ca²⁺‐channels at the plasma membrane, partly due to fewer vesicles of reduced size and synaptobrevin‐2 content. In contrast, release kinetics and Ca²⁺‐sensitivity remain unchanged, indicating that the final fusion reaction leading to transmitter release is unperturbed. Additional deletion of the closest related SNARE, vti1b, does not exacerbate the vti1a phenotype, and vti1b null cells show no secretion defects, indicating that vti1b does not participate in exocytosis. Long‐term re‐expression of vti1a (days) was necessary for restoration of secretory capacity, whereas strong short‐term expression (hours) was ineffective, consistent with vti1a involvement in an upstream step related to vesicle generation, rather than in fusion. We conclude that vti1a functions in vesicle generation and Ca²⁺‐channel trafficking, but is dispensable for transmitter release.     

Meningoencephalitis Due to Listeria monocytogenes in a Pregnant Rhesus Macaque (Macaca mulatta)

We here report a spontaneous case of meningoencephalitis due to Listeria monocytogenes in an adult primiparous rhesus macaque (Macaca mulatta) during an outbreak of listeriosis in an outdoor enclosure. Clinical signs included tremors, abnormal posture, and altered mental status. Hematology and analyses of cerebrospinal fluid were consistent with bacterial infection. Pure cultures of L. monocytogenes were recovered from the placenta–abortus, cerebrospinal fluid, and brain tissue. The macaque did not respond to treatment and was euthanized. Histopathologic examination of the brain revealed acute meningoencephalitis. This case represents an unusual clinical and pathologic presentation of listeriosis in a nonhuman primate in which the dam and fetus both were affected.Listeria monocytogenes is a ubiquitous, facultative anaerobic, intracellular gram-positive coccobacillus. This bacterium is found in diverse environments including (but not limited to) soil, water, plant matter, food items, and the intestinal tract of mammalian hosts.^15,18 The organism is environmentally resistant, being able to survive in dried media for several months and in moist soil for up to a year.¹⁵ L. monocytogenes is the causative agent of listeriosis, a bacterial infection that has a worldwide distribution and affects a wide range of mammals and birds, including human beings.In people, L. monocytogenes is a relatively uncommon foodborne pathogen; its abilities to survive food processing and grow in cold conditions allow it to persist in appropriately stored or refrigerated foods.² In people, listeriosis occurs both sporadically and as large outbreaks,¹⁸ generally comprising 3 separate syndromes with clinical manifestations ranging from mild to life-threatening.³⁵ The most common form is seen in immunocompetent, nonpregnant adults as a febrile gastroenteritis.^2,18,21 The other 2 forms, which occur in fetuses and immunocompromised patients, are more severe.^19,21 In pregnant women, maternal listeriosis is asymptomatic or causes mild, flu-like symptoms, but the bacterium''s ability to cross the placenta and the blood–brain barrier of the fetus results in neonatal septicemia, meningitis, abortion, and stillbirth.¹⁶ In elderly and immunocompromised patients, septicemia and meningoencephalitis are life-threatening manifestations of literiosis.²⁶ The worldwide case fatality rate varies widely among countries, sometimes exceeding 50% despite what is considered to be appropriate antibiotic therapy.¹⁸ In 2009, the Centers for Disease Control reported 524 cases of listeriosis in the United States, which were associated with a 19% resulting in death.⁴In ruminants, listeriosis is also known as ‘circling disease’ and ‘silage disease.’^8,18,21 Foodborne infection with L. monocytogenes is well described, and many studies have shown that spoiled silage may be a source of listeria outbreaks.^8,18 Rhombencephalitis and diffuse meningoencephalitis are the most recognized forms of the infection in ruminants; sporadic abortion is reported also.²² Clinical signs of listeria encephalitis in cattle, sheep, and goats are characterized by unilateral or bilateral brainstem dysfunction and cranial nerve deficits. In sheep and goats, the course of the disease is acute, but the disease in cattle has a more chronic progression, with neurologic manifestations that can last 4 to 14 d.^1,22In rabbits, infection with L. monocytogenes is characterized by abortion in pregnant does or sudden death; neurologic signs are rare.¹ In poultry, an acute form with septicemia and sudden death occurs in adults, in contrast to a subacute–chronic form, with encephalitis, in the young.⁶The literature on L. monocytogenes in nonhuman primates is sparse^5,11,17,33 and more recently limited to experimental infection of pregnant animals. In pregnant rhesus macaques (Macaca mulatta), experimental infection during the last trimester of gestation can cause stillbirth with no other clinical signs.^23,24 In our colony, however, infection with L. monocytogenes is endemic. Every year, several spontaneous abortions or stillbirths in our outdoor colony are caused by infection of the dam with this organism. Culture of L. monocytogenes from both the abortus–fetus and placenta are well documented. As described in the literature,^23,24 the dams in our colony do not demonstrate any clinical signs prior to the abortion or stillbirth.During the winter to spring of 2011, one of our outdoor housing enclosures experienced an outbreak of listeriosis. This outside corral housed 100 rhesus macaques in a social group that included 42 reproductive females. Of these reproductive females, 37 (88%) were confirmed pregnant by abdominal palpation or ultrasonography or both. From January 2011 to May 2011, 19 (51%) stillbirths and neonatal deaths (in infants younger than 3 d) were reported in this enclosure; 13 (68%) of these tissues (placenta, 3; fetal lungs, 8; fetal peritoneum, 2) were culture-positive for L. monocytogenes. In all cases except the one presented here, the dam did not manifest any clinical signs prior to or after the delivery of a stillborn or premature birth with neonatal death.Here we describe an unusual case of listeriosis in a primiparous pregnant female rhesus macaque that manifested severe neurologic impairment and intrautero death of the fetus.     

Retinal Vessel Width Measurement at Branchings Using an Improved Electric Field Theory-Based Graph Approach

The retinal vessel width relationship at vessel branch points in fundus images is an important biomarker of retinal and systemic disease. We propose a fully automatic method to measure the vessel widths at branch points in fundus images. The method is a graph-based method, in which a graph construction method based on electric field theory is applied which specifically deals with complex branching patterns. The vessel centerline image is used as the initial segmentation of the graph. Branching points are detected on the vessel centerline image using a set of detection kernels. Crossing points are distinguished from branch points and excluded. The electric field based graph method is applied to construct the graph. This method is inspired by the non-intersecting force lines in an electric field. At last, the method is further improved to give a consistent vessel width measurement for the whole vessel tree. The algorithm was validated on 100 artery branchings and 100 vein branchings selected from 50 fundus images by comparing with vessel width measurements from two human experts.     

Treatment with the vascular disrupting agent combretastatin is associated with impaired AQP2 trafficking and increased urine output

Combretastatin A-4 disodium phosphate (CA4P) is a vascular disrupting agent known to mediate its effects primarily on tumor blood vessels. CA4P has previously been shown to induce a significant increase in mean arterial blood pressure and in hemoglobin concentration in mice. In the present study, we examined whether this is associated with a general leakage of water into certain tissues or with changes in renal water handling. Munich-Wistar rats received either CA4P (30 mg/kg body wt) or saline intraperitoneally as a bolus injection. One hour later, hemoglobin concentration and mean blood pressure increased significantly. MRI showed no significant changes in tissue water content following CA4P administration. However, urine output and salt excretion increased 1 h after CA4P treatment, without changes in urinary and medullary osmolality. Aquaporin 2 (AQP2) mRNA levels in kidney inner medulla did not change 1 h after CA4P treatment, but semiquantitative confocal laser-scanning microscopy analysis demonstrated a decrease in phosphorylated AQP2 (pS256-AQP2) apical distribution within the collecting ducts of CA4P-treated rats compared with the characteristic apical localization in control rats. Furthermore, we demonstrated that CA4P cause disruption of microtubules and a weaker apical labeling of pS256-AQP2 in collecting duct principal cells within 1 h. In conclusion, our data indicate that water escapes from the vascular system after CA4P treatment, and it may take place primarily through a renal mechanism. The CA4P-mediated increase in urine output seems to be a local effect in the collecting ducts due to reduced AQP2 trafficking to the apical plasma membrane.     

High-throughput screen for small molecules that modulate the ATPase activity of the molecular chaperone DnaK

DnaK is a molecular chaperone of Escherichia coli that belongs to a family of conserved 70-kDa heat shock proteins. The Hsp70 chaperones are well known for their crucial roles in regulating protein homeostasis, preventing protein aggregation, and directing subcellular traffic. Given the complexity of functions, a chemical method for controlling the activities of these chaperones might provide a useful experimental tool. However, there are only a handful of Hsp70-binding molecules known. To build this area, we developed a robust, colorimetric, high-throughput screening (HTS) method in 96-well plates that reports on the ATPase activity of DnaK. Using this approach, we screened a 204-member focused library of molecules that share a dihydropyrimidine core common to known Hsp70-binding leads and uncovered seven new inhibitors. Intriguingly, the candidates do not appear to bind the hydrophobic groove that normally interacts with peptide substrates. In sum, we have developed a reliable HTS method that will likely accelerate discovery of small molecules that modulate DnaK/Hsp70 function. Moreover, because this family of chaperones has been linked to numerous diseases, this platform might be used to generate new therapeutic leads.     

Hypertonic saline reduces neutrophil-epithelial interactions in vitro and gut tissue damage in a mouse model of colitis

Transepithelial migration of polymorphonuclear neutrophils (PMN) plays a crucial role in inflammatory conditions of the intestine, such as inflammatory bowel diseases. Hypertonic saline (HS) exerts various inhibitory effects on PMN function. We hypothesized that HS could inhibit transepithelial migration of PMN and thereby prevent inflammatory events in experimental colitis. Isolated human PMN were treated with HS (40 mM), and their transmigration across a monolayer of T84 epithelial cells was induced by N-formyl-methionyl-leucyl-phenylalanine. Monolayer disruption was assessed by monitoring changes in transepithelial conductance in an Ussing chamber. Colitis in mice was induced by oral administration of dextran sulfate sodium (DSS). Animals were treated with 4 or 8 ml/kg of 7.5% saline intraperitoneally two times daily for 7 days. Controls received equivalent volumes of normal saline (NS, n = 6) or no intraperitoneal treatment (DSS, n = 12). The severity of inflammation was evaluated based on disease activity index and histology score. HS treatment of PMN in vitro significantly reduced cell migration and the disruption of T84 monolayers compared with untreated control cells (n = 5, P < 0.05). This effect of HS was dose dependent. HS treatment in vivo also reduced colitis-induced gut tissue damage, as indicated by an improved histology score compared with the NS and DSS groups. We conclude that HS inhibits transepithelial migration of PMN in vitro and gut tissue damage in vivo in a mouse model of colitis. Thus HS may have clinical value to reduce PMN-mediated intestinal damage.     

Functional variant in a bitter-taste receptor (hTAS2R16) influences risk of alcohol dependence

A coding single-nucleotide polymorphism (cSNP), K172N, in hTAS2R16, a gene encoding a taste receptor for bitter beta -glucopyranosides, shows significant association with alcohol dependence (P = .00018). This gene is located on chromosome 7q in a region reported elsewhere to exhibit linkage with alcohol dependence. The SNP is located in the putative ligand-binding domain and is associated with an increased sensitivity to many bitter beta -glucopyranosides in the presence of the N172 allele. Individuals with the ancestral allele K172 are at increased risk of alcohol dependence, regardless of ethnicity. However, this risk allele is uncommon in European Americans (minor-allele frequency [MAF] 0.6%), whereas 45% of African Americans carry the allele (MAF 26%), which makes it a much more significant risk factor in the African American population.