On the localized coupling of respiration and phosphorylation in mitochondria

This paper is an overview of the theoretical and experimental studies performed in our laboratory to answer the question whether there exist conditions where the hypothetical mechanism of the localized coupling of respiration and phosphorylation postulated by R. Williams in 1961 operates. These studies were undertaken to verify the earlier suggestion that mitochondria may exist in two structural and functional states. Correspondingly, there are two operation modes of oxidative phosphorylation, one of which corresponds to the Williams' mechanism of localized coupling and the other, to the Mitchell's mechanism of delocalized coupling. The paper considers the principle of the energy conservation of oxidative reactions in mitochondrial membranes in the form of the thermodynamic potential of hydrogen ions (Deltamusol) lacking, in part, the solvation shell. We present experimental evidence for the existence of the mechanism of localized coupling and describes the conditions favorable for its implementation. The experiments described in this paper show that the aforementioned models for proton coupling are not necessarily alternative. A conclusion is made that, depending on the particular conditions, either localized or delocalized coupling mechanisms of oxidative phosphorylation may come into operation.     

Morphogenesis as a macroscopic self-organizing process

We start from reviewing different epistemological constructions used for explaining morphogenesis. Among them, we explore the explanatory power of a law-centered approach which includes top-down causation and the basic concepts of a self-organization theory. Within such a framework, we discuss the morphomechanical models based upon the presumption of feedbacks between mechanical stresses imposed onto a given embryo part from outside and those generated within the latter as a kind of active response. A number of elementary morphogenetic events demonstrating that these feedbacks are directed towards hyper-restoration (restoration with an overshoot) of the initial state of mechanical stresses are described. Moreover, we show that these reactions are bound together into the larger scale feedbacks. That permits to suggest a reconstruction of morphogenetic successions in early Metazoan development concentrated around two main archetypes distinguished by the blastopores geometry. The perspectives of applying the same approach to cell differentiation are outlined. By discussing the problem of positional information we suggest that the developmental pathway of a given embryo part depends upon its preceded deformations and the corresponding mechanical stresses rather than upon its static position at any moment of development.     

KBTBD13 interacts with Cullin 3 to form a functional ubiquitin ligase

Bone marrow cell (BMC)-derived myofibroblast-like cells have been reported in various organs, including the pancreas. However, the contribution of these cells to pancreatic fibrosis has not been fully discussed. The present study examined the possible involvement of pancreatic stellate cells (PSCs) originating from BMCs in the development of pancreatic fibrosis in a clinically relevant rat model of acute pancreatitis induced by a choline-deficient/ethionine-supplemented (CDE) diet. BMCs from female transgenic mice ubiquitously expressing green fluorescent protein (GFP) were transplanted into lethally irradiated male rats. Once chimerism was established, acute pancreatitis was induced by a CDE diet. Chronological changes in the number of PSCs originating from the donor BMCs were examined using double immunofluorescence for GFP and markers for PSCs, such as desmin and alpha smooth muscle actin (αSMA), 1, 3 and 8 weeks after the initiation of CDE feeding. We also used immunohistochemical staining to evaluate whether the PSCs from the BMCs produce growth factors, such as platelet-derived growth factor (PDGF) and transforming growth factor (TGF) β1. The percentage of BMC-derived activated PSCs increased significantly, peaking after 1 week of CDE treatment (accounting for 23.3±0.9% of the total population of activated PSCs) and then decreasing. These cells produced both PDGF and TGFβ1 during the early stage of pancreatic fibrosis. Our results suggest that PSCs originating from BMCs contribute mainly to the early stage of pancreatic injury, at least in part, by producing growth factors in a rat CDE diet-induced pancreatitis model.     

Medieval History of the Duda’im Melon (<Emphasis Type="Italic">Cucumis melo</Emphasis>, Cucurbitaceae)

Medieval History of the Duda’im Melon ( Cucumis melo , Cucurbitaceae). Melons, Cucumis melo, are a highly polymorphic species for fruit characteristics. The melons that are the most valued are the ones that turn sweet when ripe, including the muskmelons, cantaloupes, and casabas. Others, including the elongate adzhur, conomon, and snake melons, are consumed when immature, like cucumbers. The duda’im melons, Cucumis melo Duda’im Group, are special, as their small, spherical, thin-fleshed, insipid but beautifully maroon, dark-orange, or brown-and-yellow striped ripe fruits are valued for ornament and especially for their lush fragrance. The distinctive properties of duda’im melons are matched with special names given to them in several languages and geographical areas, which have made possible tracing of the history of these melons to mid-9^th century Persia. From that region, duda’im melons diffused westward, likely facilitated by Islamic conquests, reaching North Africa and Andalusia in the 10^th century.     

Heat tolerance,temperature acclimation,acute oxidative damage and canalization of haemoglobin expression in <Emphasis Type="Italic">Daphnia</Emphasis>

Daphnia is a widespread freshwater zooplankton species, which is both a classic and emerging new model for research in ecological physiology, ecotoxicology and evolutionary biology of adaptation to novel environments. Heat tolerance in Daphnia is known to depend both upon evolutionary history of a genotype and on individuals’ acclimation to elevated temperature and to correlate with the level of haemoglobin expression. We demonstrate the existence of north–south gradient of heat tolerance in North American D. pulex, which is not associated with any parallel changes in haemoglobin expression. Geographically distinct clones differ in the way their haemoglobin expression changes due to acclimation to a sub-stressful (28°C) temperature, but these changes are not correlated with the latitude of clones’ origin. Likewise, the effect of acclimation to sub-stressful temperature is independent from, and cannot be fully explained by, haemoglobin expression changes during acclimation. The degree of oxidative damage to haemoglobin, measured as the ratio of absorbance at 540:576 nm at the acclimation temperature, is a strong predictor of 28°C-acclimated Daphnia survival during an acute heat exposure. The comparison of haemoglobin expression in resistant and tolerant clones acclimated to different temperatures indicates that tolerant clones exhibit canalization of haemoglobin expression, possessing a high level of haemoglobin even at non-stressful temperatures. We discuss the evolutionary biology of adaptation and acclimation to elevated temperatures in an ecologically important component of freshwater ecosystems in the context of global climate change.     

Haploinsufficiency of a spliceosomal GTPase encoded by EFTUD2 causes mandibulofacial dysostosis with microcephaly

Mandibulofacial dysostosis with microcephaly (MFDM) is a rare sporadic syndrome comprising craniofacial malformations, microcephaly, developmental delay, and a recognizable dysmorphic appearance. Major sequelae, including choanal atresia, sensorineural hearing loss, and cleft palate, each occur in a significant proportion of affected individuals. We present detailed clinical findings in 12 unrelated individuals with MFDM; these 12 individuals compose the largest reported cohort to date. To define the etiology of MFDM, we employed whole-exome sequencing of four unrelated affected individuals and identified heterozygous mutations or deletions of EFTUD2 in all four. Validation studies of eight additional individuals with MFDM demonstrated causative EFTUD2 mutations in all affected individuals tested. A range of EFTUD2-mutation types, including null alleles and frameshifts, is seen in MFDM, consistent with haploinsufficiency; segregation is de novo in all cases assessed to date. U5-116kD, the protein encoded by EFTUD2, is a highly conserved spliceosomal GTPase with a central regulatory role in catalytic splicing and post-splicing-complex disassembly. MFDM is the first multiple-malformation syndrome attributed to a defect of the major spliceosome. Our findings significantly extend the range of reported spliceosomal phenotypes in humans and pave the way for further investigation in related conditions such as Treacher Collins syndrome.     

The kinetics of early T and B cell immune recovery after bone marrow transplantation in RAG-2-deficient SCID patients

The kinetics of T and B cell immune recovery after bone marrow transplantation (BMT) is affected by many pre- and post-transplant factors. Because of the profoundly depleted baseline T and B cell immunity in recombination activating gene 2 (RAG-2)-deficient severe combined immunodeficiency (SCID) patients, some of these factors are eliminated, and the immune recovery after BMT can then be clearly assessed. This process was followed in ten SCID patients in parallel to their associated transplant-related complications. Early peripheral presence of T and B cells was observed in 8 and 4 patients, respectively. The latter correlated with pre-transplant conditioning therapy. Cells from these patients carried mainly signal joint DNA episomes, indicative of newly derived B and T cells. They were present before the normalization of the T cell receptor (TCR) and the B cell receptor (BCR) repertoire. Early presentation of the ordered TCR gene rearrangements after BMT occurred simultaneously, but this pattern was heterogeneous over time, suggesting different and individual thymic recovery processes. Our findings early after transplant could suggest the long-term patients' clinical outcome. Early peripheral presence of newly produced B and T lymphocytes from their production and maturation sites after BMT suggests donor stem cell origin rather than peripheral expansion, and is indicative of successful outcome. Peripheral detection of TCR excision circles and kappa-deleting recombination excision circles in RAG-2-deficient SCID post-BMT are early markers of T and B cell reconstitution, and can be used to monitor outcome and tailor specific therapy for patients undergoing BMT.     

The IAP-antagonist ARTS initiates caspase activation upstream of cytochrome C and SMAC/Diablo

ARTS (Sept4_i2) is a pro-apoptotic tumor suppressor protein that functions as an antagonist of X-linked IAP (XIAP) to promote apoptosis. It is generally thought that mitochondrial outer membrane permeabilization (MOMP) occurs before activation of caspases and is required for it. Here, we show that ARTS initiates caspase activation upstream of MOMP. In living cells, ARTS is localized to the mitochondrial outer membrane. In response to apoptotic signals, ARTS translocates rapidly to the cytosol in a caspase-independent manner, where it binds XIAP and promotes caspase activation. This translocation precedes the release of cytochrome C and SMAC/Diablo, and ARTS function is required for the normal timing of MOMP. We also show that ARTS-induced caspase activation leads to cleavage of the pro-apoptotic Bcl-2 family protein Bid, known to promote MOMP. We propose that translocation of ARTS initiates a first wave of caspase activation that can promote MOMP. This leads to the subsequent release of additional mitochondrial factors, including cytochrome C and SMAC/Diablo, which then amplifies the caspase cascade and causes apoptosis.     

VAMP-Associated Protein B (VAPB) Promotes Breast Tumor Growth by Modulation of Akt Activity

VAPB (VAMP- associated protein B) is an ER protein that regulates multiple biological functions. Although aberrant expression of VAPB is associated with breast cancer, its function in tumor cells is poorly understood. In this report, we provide evidence that VAPB regulates breast tumor cell proliferation and AKT activation. VAPB protein expression is elevated in primary and metastatic tumor specimens, and VAPB mRNA expression levels correlated negatively with patient survival in two large breast tumor datasets. Overexpression of VAPB in mammary epithelial cells increased cell growth, whereas VAPB knockdown in tumor cells inhibited cell proliferation in vitro and suppressed tumor growth in orthotopic mammary gland allografts. The growth regulation of mammary tumor cells controlled by VAPB appears to be mediated, at least in part, by modulation of AKT activity. Overexpression of VAPB in MCF10A-HER2 cells enhances phosphorylation of AKT. In contrast, knockdown of VAPB in MMTV-Neu tumor cells inhibited pAKT levels. Pharmacological inhibition of AKT significantly reduced three-dimensional spheroid growth induced by VAPB. Collectively, the genetic, functional and mechanistic analyses suggest a role of VAPB in tumor promotion in human breast cancer.     

Neuroprotective effects of N-acetyl-cysteine and acetyl-L-carnitine after spinal cord injury in adult rats

Following the initial acute stage of spinal cord injury, a cascade of cellular and inflammatory responses will lead to progressive secondary damage of the nerve tissue surrounding the primary injury site. The degeneration is manifested by loss of neurons and glial cells, demyelination and cyst formation. Injury to the mammalian spinal cord results in nearly complete failure of the severed axons to regenerate. We have previously demonstrated that the antioxidants N-acetyl-cysteine (NAC) and acetyl-L-carnitine (ALC) can attenuate retrograde neuronal degeneration after peripheral nerve and ventral root injury. The present study evaluates the effects of NAC and ALC on neuronal survival, axonal sprouting and glial cell reactions after spinal cord injury in adult rats. Tibial motoneurons in the spinal cord were pre-labeled with fluorescent tracer Fast Blue one week before lumbar L5 hemisection. Continuous intrathecal infusion of NAC (2.4 mg/day) or ALC (0.9 mg/day) was initiated immediately after spinal injury using Alzet 2002 osmotic minipumps. Neuroprotective effects of treatment were assessed by counting surviving motoneurons and by using quantitative immunohistochemistry and Western blotting for neuronal and glial cell markers 4 weeks after hemisection. Spinal cord injury induced significant loss of tibial motoneurons in L4-L6 segments. Neuronal degeneration was associated with decreased immunostaining for microtubular-associated protein-2 (MAP2) in dendritic branches, synaptophysin in presynaptic boutons and neurofilaments in nerve fibers. Immunostaining for the astroglial marker GFAP and microglial marker OX42 was increased. Treatment with NAC and ALC rescued approximately half of the motoneurons destined to die. In addition, antioxidants restored MAP2 and synaptophysin immunoreactivity. However, the perineuronal synaptophysin labeling was not recovered. Although both treatments promoted axonal sprouting, there was no effect on reactive astrocytes. In contrast, the microglial reaction was significantly attenuated. The results indicate a therapeutic potential for NAC and ALC in the early treatment of traumatic spinal cord injury.