Facultative heterochromatin: is there a distinctive molecular signature?

The Latin word "facultas" literally means "opportunity." Facultative heterochromatin (fHC) then designates genomic regions in the nucleus of a eukaryotic cell that have the opportunity to adopt open or compact conformations within temporal and spatial contexts. This review focuses on the molecular and functional aspects of fHC that distinguish it from constitutive heterochromatin (cHC) and euchromatin (EC) and discusses various concepts regarding the regulation of fHC structure. We begin by revisiting the historical developments that gave rise to our current appreciation of fHC.     

Targeting HSP70: The second potentially druggable heat shock protein and molecular chaperone?

The HSF1-mediated stress response pathway is steadily gaining momentum as a critical source of targets for cancer therapy. Key mediators of this pathway include molecular chaperones such as heat shock protein (HSP) 90. There has been considerable progress in targeting HSP90 and the preclinical efficacy and signs of early clinical activity of HSP90 inhibitors have provided proof-of-concept for targeting this group of proteins. The HSP70 family of molecular chaperones are also key mediators of the HSF-1-stress response pathway and have multiple additional roles in protein folding, trafficking and degradation, as well as regulating apoptosis. Genetic and biochemical studies have supported the discovery of HSP70 inhibitors which have the potential for use as single agents or in combination to enhance the effects of classical chemotherapeutic or molecularly targeted agents including HSP90 inhibitors. Here we provide a perspective on the progress made so far in designing agents which target the HSP70 family.     

CFTR structure and function: is there a role in the kidney?

Cystic fibrosis (CF) is a lethal autosomal recessive genetic disease caused by mutations in the CF transmembrane conductance regulator (CFTR). Mutations in the CFTR gene may result in a defective protein processing that leads to changes in function and regulation of this chloride channel. Despite of the expression of CFTR in the kidney, patients with CF do not present major renal dysfunction, but it is known that both the urinary excretion of proteins and renal capacity to concentrate and dilute urine are altered in these patients. CFTR mRNA is expressed in all nephron segments of rat and human, and this abundance is more prominent in renal cortex and outer medulla renal areas. CFTR protein was detected in apical surface of both proximal and distal tubules of rat kidney but not in the outer medullary collecting ducts. Studies have demonstrated that CFTR does not only transport Cl⁻ but also ATP. ATP transport by CFTR could be involved in the control of other ion transporters such as Na⁺ (ENaC) and K⁺ (renal outer medullary potassium) channels, especially in TAL and CCD. In the kidney, CFTR also might be involved in the endocytosis of low-molecular-weight proteins by proximal tubules. This review is focused on the CFTR function and structure, its role in the renal physiology, and its modulation by hormones involved in the control of extracellular fluid volume.     

Microtubule-bundling studies revisited: is there a role for MAPs?

The molecular mechanism of microtubule bundling has been enveloped in controversy for the past few years. At the centre of the debate are MAPs: are they necessary for the formation of microtubule bundles? In this article, Gloria Lee and Roland Brandt weigh the evidence and propose that microtubule stability might be the crucial factor in microtubule bundling. Perhaps then MAPs might act as spacer molecules between microtubules.     

Microtubule-dependent reticulopodial motility: is there a role for actin?

We summarize our recent immunocytochemical characterization of the reticulopodial cytoskeleton of two allogromiid foraminifers and our pharmacologic dissection of its motility. The reticulopodial microtubule cytoskeleton stained with an antiserum to brain microtubule-associated protein 2. Polymeric actin was localized in the reticulopodia by rhodamine-phalloidin staining. Microtubule inhibitors reversibly inhibited all aspects of motility; cytochalasins induced altered morphology and disorganization of motility but did not inhibit pseudopodial movements or intracellular transport. Simultaneous application of KCN and salicylhydroxamic acid (an alternative oxidase inhibitor) rapidly blocked all movement, indicating that motility is dependent on metabolic energy and that an alternative oxidative pathway functions in allogromiids. Micromanipulation and laser microsurgical experiments revealed tension throughout the reticulopodium. Our results suggest that microtubules are active components of the reticulopodial motile machinery. Actin may mediate substrate adhesion, whole-cell locomotion, pseudopodial tension, and coordination of the microtubule-based motility.     

Activation of development in mammals: is there a role for a sperm cytosolic factor?

In mammalian oocytes, fertilization-associated calcium [Ca2+]i oscillations are responsible for the activation of development. The mechanism(s) by which the sperm triggers the initial [Ca2+]i rise and supports long-lasting oscillations is not resolved. It has been proposed that the sperm may interact with receptors in the oocyte's plasma membrane and engage intracellular signaling pathways that result in Ca2+ release. A different line of investigation suggests that upon sperm-oocyte fusion, a sperm cytosolic factor is released into the oocyte which interacts with unknown cytosolic targets, and generates [Ca2+]i oscillations. We will discuss the most recent evidence for both lines of thought and demonstrate that injections of sperm crude extracts (SF) into mammalian oocytes trigger [Ca2+]i oscillations that support in vitro parthenogenetic development to the blastocyst stage.     

On molecular taxonomy: what is in a name?

Gene sequences of small portions of the genome are often used for premature detailed taxonomic changes, neglecting polyphasic taxonomy, which should also consider phenotypical characteristics. Three examples are given: (i) Recently, members of the genera Eperythrozoon and Haemobartonella have been moved, correctly so, from the Rickettsiales to the Mycoplasmatales, but were assigned to the genus Mycoplasma, mostly on the basis of 16S rRNA sequence analysis. Not only is the 16S rRNA sequence similarity between 'classical' Mycoplasma and these species of Eperythrozoon and Haemobartonella less than that between some other well-recognised bacterial genera, but their biological differences amply justify their classification in different genera of the Mycoplasmatales. Furthermore, the move creates considerable confusion, as it necessitates new names for some species, with more confusion likely to come when the 16S rRNA sequences of the type species of Eperythrozoon, a name which has priority over Mycoplasma, will be analysed. (ii) In the Rickettsiales, members of the genera Anaplasma, Ehrlichia, Cowdria, Neorickettsia and Wolhbachia are so closely related phylogenetically on the basis of 16S rRNA sequences, and for some also of groESL operon sequences, that they have recently been fused, correctly so, into one family, the Anaplasmataceae, while the tribes Ehrlichieae and Wolbachieae have been abolished. Sequence diversity within the 'classical' genus Ehrlichia has led to classifying E. phagocytophila (including E. equi and the agent of human granulocytic ehrlichiosis), E. platys and E. bovis in the genus Anaplasma, while others have been retained in Ehrlichia, which also includes Cowdria ruminantium. E. sennetsu and E. risticii have been transferred to the genus Neorickettsia. 16S rRNA and GroEL sequences of 'classical' Anaplasma and some members of 'classical' Ehrlichia do show a close relationship, but differences in citrate synthase gene sequences, the GC content of this gene, and sequences of the gene encoding the beta-subunit of RNA polymerase, not to speak of the phenotypical differences, do not justify the fusion into one genus. Because of the phylogenetical diversity in Ehrlichia it is recommended that a new genus name be created for the E. phagocytophila genogroup (and E. platys and E. bovis). (iii) One of the conclusions of studies on the phylogeny of ticks of the subfamilies Rhipicephalinae and Hyalomminae, based on nucleotide sequences from 12S rRNA, cytochrome c oxidase I, the internal transcribed spacer 2, 18S rRNA, as well as morphological characters, is that Boophilus should be considered as a subgenus of Rhipicephalus. While Boophilus and Rhipicephalus are undoubtedly close, the obviously important morphological and biological differences between the genera Rhipicephalus and Boophilus are thus overruled by similarities in the sequences of a number of genes and this leads to considerable confusion. Polyphasic taxonomy amply justifies maintaining Boophilus as a separate genus, phylogenetically near to Rhipicephalus. This note is a plea for a cautious and balanced approach to taxonomy, taking into account molecular genotypical information, as far as is possible from different genes, as well as phenotypical characteristics.     

Response facilitation in the four great apes: is there a role for empathy?

Contagious yawning is a form of response facilitation found in humans and other primates in which observing a model yawning enhances the chance that the observer will also yawn. Because contagious yawning seems to be more easily triggered when models are conspecifics or have a strong social bond with the observer, it has been proposed that contagious yawning is linked to empathy. A possible way to test this hypothesis is to analyze whether individuals’ responses differ when they observe models yawning or performing different involuntary (i.e., nose wiping, scratching) and voluntary (i.e., hand closing, wrist shaking) actions that are not linked to empathy. In this study, we tested the four great ape species with two different setups by exposing them to a human experimenter repeatedly performing these actions online, and video-recorded conspecifics repeatedly performing these actions on a screen. We examined which behaviors were subject to response facilitation, whether response facilitation was triggered by both human models and video-recorded conspecifics, and whether all species showed evidence of response facilitation. Our results showed that chimpanzees yawned significantly more when and shortly after watching videos of conspecifics (but not humans) yawning than in control conditions, and they did not do so as a response to increased levels of anxiety. For all other behaviors, no species produced more target actions when being exposed to either model than under control conditions. Moreover, the individuals that were more “reactive” when watching yawning videos were not more reactive when exposed to other actions. Since, at least in chimpanzees, (1) subjects only showed response facilitation when they were exposed to yawning and (2) only if models were conspecifics, it appears that contagious yawning is triggered by unique mechanisms and might be linked to empathy.     

Preventing α-synuclein aggregation: The role of the small heat-shock molecular chaperone proteins

Protein homeostasis, or proteostasis, is the process of maintaining the conformational and functional integrity of the proteome. The failure of proteostasis can result in the accumulation of non-native proteins leading to their aggregation and deposition in cells and in tissues. The amyloid fibrillar aggregation of the protein α-synuclein into Lewy bodies and Lewy neuritis is associated with neurodegenerative diseases classified as α-synucleinopathies, which include Parkinson's disease and dementia with Lewy bodies. The small heat-shock proteins (sHsps) are molecular chaperones that are one of the cell's first lines of defence against protein aggregation. They act to stabilise partially folded protein intermediates, in an ATP-independent manner, to maintain cellular proteostasis under stress conditions. Thus, the sHsps appear ideally suited to protect against α-synuclein aggregation, yet these fail to do so in the context of the α-synucleinopathies. This review discusses how sHsps interact with α-synuclein to prevent its aggregation and, in doing so, highlights the multi-faceted nature of the mechanisms used by sHsps to prevent the fibrillar aggregation of proteins. It also examines what factors may contribute to α-synuclein escaping the sHsp chaperones in the context of the α-synucleinopathies.     

Hydroxyapatite ceramics in multilevel cervical interbody fusion - is there a role?

The aim of this study is to evaluate the efficacy of hydroxyapatite grafts in multilevel cervical interbody fusion during the one year follow-up. A total of 86 patients with degenerative cervical disc disease underwent all together 224 cervical interbody fusion procedures in which either Smith-Robinson or Cloward type hydroxyapatite grafts were used. The surgeries included radiculopathy in 38 cases, myelopathy in 20 cases and myeloradicuopathy in 28 patients. In 65 out of 86 patients, fusion was followed by an anterior instrumentation (plating). Postoperatively, patients were followed for a mean of 15.64 (range 11-23.3) months. All patients underwent radiography to evaluate fusion and the axis curvature. Excellent clinical results (86%), described as a complete or partial relief of symptoms with full return to preop activity, were obtained in patients with radiculopathy. There were 5 grafts mobilizations and one graft fracture. Two grafts extruded in non-instrumented patients and required repeated surgery. There were other three reoperations due to the hardware problems. One year fusion rate was obtained at 86% for two-level surgery, 80.1% for three-level surgery and 74% for four-level surgery. The mean (SD) hospital stay was 3.8 (0.7) days. A hydroxyapatite cheramic can be a very effective synthetic material for multilevel cervical interbody fusion. It is characterized by a high fusion rate and a small percentage of graft-related complications, especially when fusion procedure is followed by plating.     

Stem cells: is there a future in plastics?

The concept that ostensibly tissue-specific stem cells can give rise to cells of heterologous lineages has gained support from studies using purified hematopoietic stem cells and sensitive donor-cell tracking methods. The ability to exploit these findings in clinical settings will probably depend on new insights into the mechanisms by which such stem cells or their progeny migrate to sites of organ damage and differentiate to cell types competent to participate in tissue regeneration.     

Is protein disulfide isomerase a redox-dependent molecular chaperone?

Protein disulfide isomerase (PDI) is a multifunctional protein catalysing the formation of disulfide bonds, acting as a molecular chaperone and being a component of the enzymes prolyl 4-hydroxylase (P4H) and microsomal triglyceride transfer protein. The role of PDI as a molecular chaperone or polypeptide-binding protein is mediated primarily through an interaction of substrates with its b' domain. It has been suggested that this binding is regulated by the redox state of PDI, with association requiring the presence of glutathione, and dissociation the presence of glutathione disulfide. To determine whether this is the case, we investigated the ability of PDI to bind to a folding polypeptide chain within a functionally intact endoplasmic reticulum and to be dissociated from the alpha-subunit of P4H in vitro in the presence of reducing or oxidizing agents. Our results clearly demonstrate that binding of PDI to these polypeptides is not regulated by its redox state. We also demonstrate that the dissociation of PDI from substrates observed in the presence of glutathione disulfide can be explained by competition for the peptide-binding site on PDI.     

Cardiac effects of oxytocin: is there a role for this peptide in cardiovascular homeostasis?

Oxytocin is well known for its role in reproduction. However, evidence has emerged suggesting a role in cardiovascular and hydroelectrolytic homeostasis. Although its renal effects have been characterized, the cardiac ones have not been much studied. Therefore, we aimed to investigate the cardiac effects of oxytocin both in vivo and in vitro. In unanesthetized rats (n=6) intravenous oxytocin (1 mug) decreased dP/dt(max) by 15% (P<0.05) and heart rate by 20% (P<0.001), at the first minute after injection. dP/dt(max) was still lower in OT-treated rats than in controls (n=8) after 15 min (P<0.05), while heart rate returned to control values after 5 min. In isolated hearts, oxytocin was able to promote negative inotropic and chronotropic effects. Perfusion with 10(-5), 10(-6) and 10(-7)M oxytocin resulted in approximately 60% (P<0.01), 25% (P<0.01) and 10% (P<0.05) reduction of left ventricle developed pressure, without effect in lower concentrations (10(-10) to 10(-8) M). Also, dP/dt(max) was reduced by 45 and 20% (10(-5) e 10(-6) M; P<0.01), while diastolic pressure raised and heart rate fell only with 10(-5)M oxytocin (P<0.05). Intravenous oxytocin (1 mug; n=6) increased arterial pressure by 22% at the first minute (+23+/-3 mm Hg; P<0.001), returning to control value thereafter. Thus, oxytocin is able to promote directly negative inotropic and chronotropic effects, but its in vivo effect also involves a reflex mechanism, originated from its pressor effect.     

The cytoskeleton in plasmodesmata: a role in intercellular transport?

Actin and myosin are components of the plant cell cytoskeleton that extend from cell to cell through plasmodesmata (PD), but it is unclear how they are organized within the cytoplasmic sleeve or how they might behave as regulatory elements. Early work used antibodies to locate actin and myosin to PD, at the electron microscope level, or to pitfields (aggregations of PD in the cell wall), using immunofluorescence techniques. More recently, a green fluorescent protein (GFP)-tagged plant myosin VIII was located specifically at PD-rich pitfields in cell walls. Application of actin or myosin disrupters may modify the conformation of PD and alter rates of cell-cell transport, providing evidence for a role in regulating PD permeability. Intriguingly, there is now evidence of differentiation between types of PD, some of which open in response to both actin and myosin disrupters, and others which are unaffected by actin disrupters or which close in response to myosin inhibitors. Viruses also interact with elements of the cytoskeleton for both intracellular and intercellular transport. The precise function of the cytoskeleton in PD may change during cell development, and may not be identical in all tissue types, or even in all PD within a single cell. Nevertheless, it is likely that actin- and myosin-associated proteins play a key role in regulating cell-cell transport, by interacting with cargo and loading it into PD, and may underlie the capacity for one-way transport across particular cell and tissue boundaries.     

Metabolism and longevity: is there a role for membrane fatty acids?

More than 100 years ago, Max Rubner combined the fact that both metabolic rate and longevity of mammals varies with body size to calculate that "life energy potential" (lifetime energy turnover per kilogram) was relatively constant. This calculation linked longevity to aerobic metabolism which in turn led to the "rate-of-living" and ultimately the "oxidative stress" theories of aging. However, the link between metabolic rate and longevity is imperfect. Although unknown in Rubner's time, one aspect of body composition of mammals also varies with body size, namely the fatty acid composition of membranes. Fatty acids vary dramatically in their susceptibility to peroxidation and the products of lipid peroxidation are very powerful reactive molecules that damage other cellular molecules. The "membrane pacemaker" modification of the "oxidative stress" theory of aging proposes that fatty acid composition of membranes, via its influence on peroxidation of lipids, is an important determinant of lifespan (and a link between metabolism and longevity). The relationship between membrane fatty acid composition and longevity is discussed for (1) mammals of different body size, (2) birds of different body size, (3) mammals and birds that are exceptionally long-living for their size, (4) strains of mice that vary in longevity, (5) calorie-restriction extension of longevity in rodents, (6) differences in longevity between queen and worker honeybees, and (7) variation in longevity among humans. Most of these comparisons support an important role for membrane fatty acid composition in the determination of longevity. It is apparent that membrane composition is regulated for each species. Provided the diet is not deficient in polyunsaturated fat, it has minimal influence on a species' membrane fatty acid composition and likely also on it's maximum longevity. The exceptional longevity of Homo sapiens combined with the limited knowledge of the fatty acid composition of human tissues support the potential importance of mitochondrial membranes in determination of longevity.