Johannes J. L. van der Want

Dynamic changes in the localization of thermally unfolded nuclear proteins associated with chaperone-dependent protection

Molecular chaperones are involved in the protection of cells against protein damage through their ability to hold, disaggregate, and refold damaged proteins or their ability to facilitate degradation of damaged proteins. Little is known about how these processes are spatially coordinated in cells. Using a heat-sensitive nuclear model protein luciferase fused to the traceable, heat-stable enhanced green fluorescent protein (N-luc-EGFP), we now show that heat inactivation and insolubilization of luciferase were associated with accumulation of N-luc-EGFP at multiple foci throughout the nucleus. Coexpression of Hsp70, one of the major mammalian chaperones, reduced the formation of these small foci during heat shock. Instead, the heat-unfolded N-luc-EGFP accumulated in large, insoluble foci. Immunofluorescence analysis revealed that these foci colocalized with the nucleoli. Time-lapse analysis demonstrated that protein translocation to the nucleolus, in contrast to the accumulation at small foci, was fully reversible upon return to the normal growth temperature. This reversibility was associated with an increase in the level of active and soluble luciferase. Expression of a carboxyl-terminal deletion mutant of Hsp70(1–543), which lacked chaperone activity, had no effect on the localization of N-luc-EGFP, which suggests that the Hsp70 chaperone activity is required for the translocation events. Our data show that Hsp70 not only is involved in holding and refolding of heat-unfolded nuclear proteins but also drives them to the nucleolus during stress. This might prevent random aggregation of thermolabile proteins within the nucleus, thereby allowing their refolding at the permissive conditions and preventing indirect damage to other nuclear components.

Prolonged cigarette smoke exposure alters mitochondrial structure and function in airway epithelial cells

BackgroundCigarette smoking is the major risk factor for COPD, leading to chronic airway inflammation. We hypothesized that cigarette smoke induces structural and functional changes of airway epithelial mitochondria, with important implications for lung inflammation and COPD pathogenesis.MethodsWe studied changes in mitochondrial morphology and in expression of markers for mitochondrial capacity, damage/biogenesis and fission/fusion in the human bronchial epithelial cell line BEAS-2B upon 6-months from ex-smoking COPD GOLD stage IV patients to age-matched smoking and never-smoking controls.ResultsWe observed that long-term CSE exposure induces robust changes in mitochondrial structure, including fragmentation, branching and quantity of cristae. The majority of these changes were persistent upon CSE depletion. Furthermore, long-term CSE exposure significantly increased the expression of specific fission/fusion markers (Fis1, Mfn1, Mfn2, Drp1 and Opa1), oxidative phosphorylation (OXPHOS) proteins (Complex II, III and V), and oxidative stress (Mn-SOD) markers. These changes were accompanied by increased levels of the pro-inflammatory mediators IL-6, IL-8, and IL-1β. Importantly, COPD primary bronchial epithelial cells (PBECs) displayed similar changes in mitochondrial morphology as observed in long-term CSE-exposure BEAS-2B cells. Moreover, expression of specific OXPHOS proteins was higher in PBECs from COPD patients than control smokers, as was the expression of mitochondrial stress marker PINK1.ConclusionThe observed mitochondrial changes in COPD epithelium are potentially the consequence of long-term exposure to cigarette smoke, leading to impaired mitochondrial function and may play a role in the pathogenesis of COPD.

Pantethine rescues a Drosophila model for pantothenate kinase-associated neurodegeneration

Pantothenate kinase–associated neurodegeneration (PKAN), a progressive neurodegenerative disorder, is associated with impairment of pantothenate kinase function. Pantothenate kinase is the first enzyme required for de novo synthesis of CoA, an essential metabolic cofactor. The pathophysiology of PKAN is not understood, and there is no cure to halt or reverse the symptoms of this devastating disease. Recently, we and others presented a PKAN Drosophila model, and we demonstrated that impaired function of pantothenate kinase induces a neurodegenerative phenotype and a reduced lifespan. We have explored this Drosophila model further and have demonstrated that impairment of pantothenate kinase is associated with decreased levels of CoA, mitochondrial dysfunction, and increased protein oxidation. Furthermore, we searched for compounds that can rescue pertinent phenotypes of the Drosophila PKAN model and identified pantethine. Pantethine feeding restores CoA levels, improves mitochondrial function, rescues brain degeneration, enhances locomotor abilities, and increases lifespan. We show evidence for the presence of a de novo CoA biosynthesis pathway in which pantethine is used as a precursor compound. Importantly, this pathway is effective in the presence of disrupted pantothenate kinase function. Our data suggest that pantethine may serve as a starting point to develop a possible treatment for PKAN.

Nonpenetrating vascular clips for small-caliber anastomosis

In the search for better anastomosing techniques, an improved vascular stapler device (VCS clip applier system®) has been introduced. The system uses nonpenetrating clips to approximate everted vessel walls. The objective of this study was to determine the effects of nonpenetrating vascular clips on endothelial wound healing. Aortic end‐to‐end anastomoses were performed in male Wistar rats. A comparison was made between clipped (n = 12) and conventional hand‐sewn (n = 6) anastomoses. Patency rates were verified at different time intervals (after 1, 4, and 8 weeks), after which the anastomotic sites were removed. Morphological evaluation was carried out using scanning electron microscopy. All rats survived the procedure. Closure with clips took less time than closure with conventional sutures, with decreasing aortic clamping times for the clipped procedures during the course of the experiments. Patency rates were 100% in both the “clipped” and “sutured” groups. Microscopic examination showed favorable endothelial healing at the clipped anastomotic sites, with less inflammatory reaction at 1 week, and a more complete endothelial regeneration at 4 and 8 weeks follow‐up, as compared with the sutured anastomoses. The clip applier holds the promise of a useful device in anastomosing small‐caliber vessels, since clip closure takes less time than suturing, while patency rates are identical, and morphological results are favorable. Training is mandatory to obtain technical skills and to achieve optimal results.

Lens epithelial cell layer formation related to hydrogel foldable intraocular lenses

PURPOSE To determine the incidence of postoperative lens epithelial cell (LEC) layer formation anterior to the Hydroview hydrogel foldable intraocular lens (IOL), the effect on vision, and the appearance under scanning electron microscopy (SEM). SETTING Eye Unit of a district general hospital, Delfzijl, and the Laboratory for Cell Biology and Electron Microscopy, Faculty of Medical Sciences, University of Groningen, The Netherlands. METHODS In the prospective study, 207 eyes that received a Hydroview hydrogel foldable IOL during 1996 were followed for almost 2 years (range 8 to 98 weeks). Eleven eyes had follow-up of fewer than 8 weeks and were excluded. Some eyes had a transparent to milky-white membrane anterior to the IOL in the plane of capsulorhexis. In the absence of other contributing factors, if best corrected visual acuity was worse than expected, it was assumed to be caused by the membrane. Depending on the membranes thickness, determined at the slitlamp, patients were offered a neodymium:YAG (Nd:YAG) laser or surgical membranectomy. If surgically excised, the layer was studied by SEM. RESULTS Of the 196 eyes, 62 eyes (33.16%) developed a thin epithelial cell layer. The membrane appeared a mean of 38.5 weeks postoperatively (range 9.9 to 85.6 weeks). Eleven eyes (5.61%) had visual symptoms varying from low vision to hazy vision or light scatter. Visual symptoms were present even in cases in which the visual axis was not invaded by the membrane. Once this layer was removed surgically or by the Nd:YAG laser, vision improved and symptoms were relieved. Four eyes (2.04%) required a surgical membranectomy. Examination of these removed membranes by SEM showed their multilayer nature and that the LECs had differentiated into fibroblast-like cells with collagenous fibrils and extracellular matrix. CONCLUSIONS The incidence of LEC membrane formation was higher than expected and increased with time in eyes with a Hydroview IOL. The membrane caused visual symptoms by occluding the visual axis, causing striation on the membrane and the IOL to decenter, tilt, or fold. These symptoms improved after the membrane was removed.

Magnetic minerals. Keystone-like crystals in cells of hornet combs.

The hexagonal brood-rearing cells inside the nest combs of the hornet Vespa orientalis are uniform in both their architecture and orientation. We have discovered that each cell contains a minute crystal that projects down from the centre of its domed roof and has a composition typical of the magnetic mineral ilmenite. These tiny crystals form a network that may act like a surveyors spirit-level, helping the hornets to assess the symmetry and balance of the cells and the direction of gravity while they are building the comb.

Lipid droplets hypertrophy: a crucial determining factor in insulin regulation by adipocytes

Lipid droplets (LDs) hypertrophy in adipocytes is the main cause of energy metabolic system dysfunction, obesity and its afflictions such as T2D. However, the role of adipocytes in linking energy metabolic disorders with insulin regulation is unknown in humans. Human adipocytes constitutively synthesize and secrete insulin, which is biologically functional. Insulin concentrations and release are fat mass- and LDs-dependent respectively. Fat reduction mediated by bariatric surgery repairs obesity-associated T2D. The expression of genes, like PCSK1 (proinsulin conversion enzyme), GCG (Glucagon), GPLD1, CD38 and NNAT, involved in insulin regulation/release were differentially expressed in pancreas and adipose tissue (AT). INS (insulin) and GCG expression reduced in human AT-T2D as compared to AT-control, but remained unchanged in pancreas in either state. Insulin levels (mRNA/protein) were higher in AT derived from prediabetes BB rats with destructed pancreatic β-cells and controls than pancreas derived from the same rats respectively. Insulin expression in 10 human primary cell types including adipocytes and macrophages is an evidence for extrapancreatic insulin-producing cells. The data suggest a crosstalk between AT and pancreas to fine-tune energy metabolic system or may minimize the metabolic damage during diabetes. This study opens new avenues towards T2D therapy with a great impact on public health.

A discrete dopaminergic projection from the incertohypothalamic A13 cell group to the dorsolateral periaqueductal gray in rat

Several findings have indicated an involvement of dopamine in panic and defensive behaviors. The dorsolateral column of the periaqueductal gray (dlPAG) is crucially involved in the expression of panic attacks in humans and defensive behaviors, also referred to as panic-like behaviors, in animals. Although the dlPAG is known to receive a specific innervation of dopaminergic fibers and abundantly expresses dopamine receptors, the origin of this dopaminergic input is largely unknown. This study aimed at mapping the dopaminergic projections to the dlPAG in order to provide further insight into the panic-like related behavior circuitry of the dlPAG. For this purpose, the retrograde tracer cholera toxin subunit b (CTb) was injected into the dlPAG of male Wistar rats and double immunofluorescence for CTb and tyrosine hydroxylase (TH), the rate-limiting enzyme in the synthesis of dopamine, was performed. Neurons labeled for both CTb and TH were counted in different dopaminergic cell groups. The findings indicate that the dopaminergic nerve terminals present in the dlPAG originate from multiple dopamine-containing cell groups in the hypothalamus and mesencephalon. Interestingly, the A13 cell group is the main source of dopaminergic afferents to the dlPAG and contains at least 45% of the total number of CTb/TH-positive neurons. Anterograde tracing with biotinylated dextran amine (BDA) combined with double immunofluorescence for BDA and TH confirmed the projections from the A13 cell group to the dlPAG. The remainder of the dopamine-positive terminals present in the dlPAG was found to originate from the extended A10 cell group and the A11 group. The A13 cell group is known to send dopaminergic efferents to several other brain regions implicated in defensive behavior, including the central amygdala and ventromedial hypothalamus. Therefore, although direct behavioral evidence is lacking, our finding that the A13 cell group is also the main source of dopaminergic input to the dlPAG suggests that dopamine might contribute to the regulation of dlPAG-mediated defensive behaviors.

Early hypergravity exposure effects calbindin-D28k and inositol-3-phosphate expression in Purkinje cells

In this study the effects of hypergravity were analyzed on cerebellar Purkinje cells during early development in rats. The cerebellum is a key structure in the control and the adaptation of posture and anti-gravity activities. This holds particularly when external conditions are modified. Three groups of rats were conceived, born and reared in hypergravity (2g). At postnatal day 5 (P5), P10 or P15, they were exposed to normal gravity and at P40, the cerebella were investigated on the expression of calbindin-D28k and inositol-3-phosphate (IP3) in Purkinje cells. Control animals were bred in the same conditions but at 1g. Immunoreactivity of Purkinje cells was studied in lobules III and IX of the vermis. Lobule IX of the vermis is one of the targets of primary otolithic vestibular projections, and lobule III served as a control, being much less related with vestibular inputs. The results show that hypergravity induces a decrease in calbindin and IP3 labeling in 20% of Purkinje cells of lobule IX without any change in lobule III. Animals transferred from 2g to 1g at P5 or P10 showed the most pronounced effects and much less at P15. This study demonstrates that early development of the cerebellum is highly sensitive to changes in gravity. Ages until P10 are critical for the development of vestibulo-cerebellar connections, and in particularly the calcium signaling in Purkinje cells.

CRF and urocortin differentially modulate GluRδ2 expression and distribution in parallel fiber–Purkinje cell synapses

Corticotropin-releasing factor (CRF) and urocortin (UCN) are closely related multifunctional regulators, governing, among other processes, Purkinje cell development. Here, we investigate the effects of CRF and UCN on Purkinje cells in organotypic slices. We show that both peptides upregulate delta2 ionotropic glutamate receptor gene expression, and increase the abundance of the receptor in the postsynaptic density. However, only UCN treatment results in increased delta2 protein level per Purkinje cell, implying the existence of posttranscriptional regulation of GluRdelta2 mRNA. CRF, in contrast, reduces the number of delta2-positive dendritic shafts per cell, implying that the increase of GluRdelta2 in remaining synapses may be mainly due to its retargeting. We further observed different patterns of GluRdelta2 distribution in the zone of postsynaptic density upon CRF and UCN treatment. CRF treatment results in a clustered distribution of GluRdelta2 along the postsynaptic density, whereas UCN treatment provides a linear distribution.