Disturbed neurotransmitter homeostasis in ether lipid deficiency.

Plasmalogens, the most prominent ether (phospho)lipids in mammals, are structural components of most cellular membranes. Due to their physicochemical properties and abundance in the central nervous system, a role of plasmalogens in neurotransmission has been proposed, but conclusive data are lacking. Here, we targeted this issue in the glyceronephosphate O-acyltransferase (Gnpat) KO mouse, a model of complete deficiency in ether lipid biosynthesis. Throughout the study, focusing on adult male animals, we found reduced brain levels of various neurotransmitters. In the dopaminergic nigrostriatal tract, synaptic endings but not neuronal cell bodies were affected. Neurotransmitter turnover was altered in ether lipid-deficient murine as well as human post-mortem brain tissue. A generalized loss of synapses did not account for the neurotransmitter deficits, since the levels of several presynaptic proteins appeared unchanged. However, reduced amounts of vesicular monoamine transporter indicate a compromised vesicular uptake of neurotransmitters. As exemplified by norepinephrine, the release of neurotransmitters from Gnpat KO brain slices was diminished in response to strong electrical and chemical stimuli. Finally, addressing potential phenotypic correlates of the disturbed neurotransmitter homeostasis, we show that ether lipid deficiency manifests as hyperactivity and impaired social interaction. We propose that the lack of ether lipids alters the properties of synaptic vesicles leading to reduced amounts and release of neurotransmitters. These features likely contribute to the behavioral phenotype of Gnpat KO mice, potentially modeling some human neurodevelopmental disorders like autism or attention deficit hyperactivity disorder.

Small GTPases in peroxisome dynamics.

In this review article, we summarize current knowledge on peroxisome biogenesis/functions and the role that small GTPases may play in these processes. Precise intracellular distribution of cell organelles requires their regulated association to microtubules and the actin cytoskeleton. In this respect, RhoGDP/RhoGTP favor binding of peroxisomes to microtubules and actin filaments. In its GTP-bound form, RhoA activates a regulatory cascade involving Rho kinaseII and non-muscle myosinIIA. Such interactions frequently depend on phosphoinositides (PIs) of which PI4P, PI(4,5)P2, and PI(3,5)P2 were found to be present in the peroxisomal membrane. PIs are pivotal determinants of intracellular signaling and known to regulate a wide range of cellular functions. In many of these functions, small GTPases are implicated. The small GTPase ADP-ribosylation factor 1 (Arf1), for example, is known to stimulate synthesis of PI4P and PI(4,5)P2 on the Golgi to regulate protein and lipid sorting. In vitro binding assays localized Arf1 and the COPI complex to peroxisomes. In light of the recent discussion of pre-peroxisomal vesicle generation at the ER, peroxisomal Arf1-COPI vesicles may serve retrograde transport of ER-resident components. A mass spectrometric screen localized various Rab proteins to peroxisomes. Overexpression of these proteins in combination with laser-scanning fluorescence microscopy co-localized Rab6, Rab8, Rab10, Rab14, and Rab18 with peroxisomal structures. By analogy to the role these proteins play in other organelle dynamics, we may envisage what the function of these proteins may be in relation to the peroxisomal compartment.

Homeostasis of phospholipids - The level of phosphatidylethanolamine tightly adapts to changes in ethanolamine plasmalogens.

Ethanolamine plasmalogens constitute a group of ether glycerophospholipids that, due to their unique biophysical and biochemical properties, are essential components of mammalian cellular membranes. Their importance is emphasized by the consequences of defects in plasmalogen biosynthesis, which in humans cause the fatal disease rhizomelic chondrodysplasia punctata (RCDP). In the present lipidomic study, we used fibroblasts derived from RCDP patients, as well as brain tissue from plasmalogen-deficient mice, to examine the compensatory mechanisms of lipid homeostasis in response to plasmalogen deficiency. Our results show that phosphatidylethanolamine (PE), a diacyl glycerophospholipid, which like ethanolamine plasmalogens carries the head group ethanolamine, is the main player in the adaptation to plasmalogen insufficiency. PE levels were tightly adjusted to the amount of ethanolamine plasmalogens so that their combined levels were kept constant. Similarly, the total amount of polyunsaturated fatty acids (PUFAs) in ethanolamine phospholipids was maintained upon plasmalogen deficiency. However, we found an increased incorporation of arachidonic acid at the expense of docosahexaenoic acid in the PE fraction of plasmalogen-deficient tissues. These data show that under conditions of reduced plasmalogen levels, the amount of total ethanolamine phospholipids is precisely maintained by a rise in PE. At the same time, a shift in the ratio between ω-6 and ω-3 PUFAs occurs, which might have unfavorable, long-term biological consequences. Therefore, our findings are not only of interest for RCDP but may have more widespread implications also for other disease conditions, as for example Alzheimer's disease, that have been associated with a decline in plasmalogens.

Impaired neurotransmission in ether lipid-deficient nerve terminals.

Isolated defects of ether lipid (EL) biosynthesis in humans cause rhizomelic chondrodysplasia punctata type 2 and type 3, serious peroxisomal disorders. Using a previously described mouse model [Rodemer, C., Thai, T.P., Brugger, B., Kaercher, T., Werner, H., Nave, K.A., Wieland, F., Gorgas, K., and Just, W.W. (2003) Inactivation of ether lipid biosynthesis causes male infertility, defects in eye development and optic nerve hypoplasia in mice. Hum. Mol. Genet., 12, 1881-1895], we investigated the effect of EL deficiency in isolated murine nerve terminals (synaptosomes) on the pre-synaptic release of the neurotransmitters (NTs) glutamate and acetylcholine. Both Ca(2+)-dependent exocytosis and Ca(2+)-independent efflux of the transmitters were affected. EL-deficient synaptosomes respire at a reduced rate and exhibit a lowered adenosin-5'-triphosphate/adenosine diphosphate (ATP/ADP) ratio. Consequently, ATP-driven processes, such as synaptic vesicle cycling and maintenance of Na(+), K(+) and Ca(2+) homeostasis, might be disturbed. Analyzing reactive oxygen species in EL-deficient neural and non-neural tissues revealed that plasmalogens (PLs), the most abundant EL species in mammalian central nervous system, considerably contribute to the generation of the lipid peroxidation product malondialdehyde. Although EL-deficient tissue contains less lipid peroxidation products, fibroblasts lacking ELs are more susceptible to induced oxidative stress. In summary, these results suggest that due to the reduced energy state of EL-deficient tissue, the Ca(2+)-independent efflux of NTs increases while the Ca(2+)-dependent release declines. Furthermore, lack of PLs is mainly compensated for by an increase in the concentration of phosphatidylethanolamine and results in a significantly lowered level of lipid peroxidation products in the brain cortex and cerebellum.

[General anesthesia for ambulatory surgery : Clinical pharmacological considerations on the practical approach]. / Allgemeinanästhesie bei ambulanten Operationen : Klinisch-pharmakologische Überlegungen zum praktischen Vorgehen.

Due to modern surgical and anesthesia techniques, many patients undergoing small or even medium surgical procedures will recover within minutes and can then be discharged after a few hours of monitoring. Aside from an optimized surgical technique, a precise and differentiated anesthesia concept is needed to guarantee rapid recovery and home readiness. Nowadays, remifentanil-propofol represents the standard regime in ambulatory anesthesia. The use of alfentanil, desfluran or sevofluran is also possible whereas other intravenous or inhaled anesthetics or other opioids are rarely used. If endotracheal intubation is necessary, a reduced intubating dose of neuromuscular blockers (NMB), such as mivacurium, atracurium and rocuronium, i.e. 1-1.5-times the 95 % effective dose (ED95) is a good possibility to accelerate neuromuscular recovery while still having acceptable intubation conditions. Due to its limitations and contraindications, succinylcholine is not the first choice but may be used in non-fasting patients in need of urgent (ambulatory) surgery, e.g. in bleeding women undergoing dilation and curettage. Even with these reduced dosages monitoring of neuromuscular recovery is crucial and should be applied to all patients when NMBs are used. Furthermore, patients should receive a risk-adapted postoperative nausea and vomiting (PONV) prophylaxis, e.g. with 4 mg dexamethasone and 4 mg ondansetron. Postdischarge nausea and vomiting (PDNV) should be anticipated by a new risk score and prophylaxis or treatment should be initiated. For postoperative pain relief, local or regional anesthesia techniques, such as infiltration, field or nerve blocks should be applied where possible. In addition, non-opioid analgesics are the basic treatment while longer-lasting opioids are only necessary for some patients.

Clinical features of critically ill patients with Shiga toxin-induced hemolytic uremic syndrome.

OBJECTIVE: In Spring 2011, an unprecedented outbreak of Shiga toxin-producing Escherichia coli serotype O104:H4-associated hemolytic uremic syndrome occurred in Northern Germany. The aim of this study was to describe the clinical characteristics, treatments, and outcomes of critically ill patients with Shiga toxin-producing E. coli-associated hemolytic uremic syndrome during this outbreak. DESIGN, SETTING, AND PATIENTS: Multicenter, retrospective, observational study of critically ill adult patients with Shiga toxin-producing E. coli-associated hemolytic uremic syndrome in six hospitals in Hamburg, Germany, between May 2011 and August 2011. MEASUREMENTS AND MAIN RESULTS: During the study period, 106 patients with Shiga toxin-producing E. coli-associated hemolytic uremic syndrome were admitted to eight ICUs. The median age was 40 years (range, 18-83) with a female:male ratio of 3:1. The median time from onset of clinical symptoms to hospital admission was 3 days and from hospital to ICU admission an additional 3 days. A total of 101 patients (95.3%) had acute renal failure and 78 (73.6%) required renal replacement therapy. Intubation and mechanical ventilation were required in 38 patients (35.8%) and noninvasive ventilation was required in 17 patients (16.0%). The median duration of invasive ventilation was 7 days (range, 1-32 days) and the median ICU stay was 10 days (range, 1-45 days). Fifty-one patients (48.1%) developed sepsis; of these 51 patients, 27 (25.4%) developed septic shock. Seventy patients (66.0%) developed severe neurological symptoms. Ninety-seven patients (91.5%) were treated with plasma exchange and 50 patients (47.2%) received eculizumab (monoclonal anti-C5 antibody). The mortality rate was 4.7%. Mild residual neurological symptoms were present in 21.7% of patients at ICU discharge, and no patient required renal replacement therapy 6 months after ICU admission. CONCLUSIONS: During the 2011 Shiga toxin-producing E. coli-associated hemolytic uremic syndrome outbreak in Germany, critical illness developed rapidly after hospital admission, often in young women. The infection was associated with severe neurological and renal symptoms, requiring mechanical ventilation and renal replacement therapy in a substantial proportion of patients. Overall, recovery was much better than expected.

The phosphoinositide 3-kinase Vps34p is required for pexophagy in Saccharomyces cerevisiae.

PIds (phosphoinositides) are phosphorylated derivatives of the membrane phospholipid PtdIns that have emerged as key regulators of many aspects of cellular physiology. We have discovered a PtdIns3P-synthesizing activity in peroxisomes of Saccharomyces cerevisiae and have demonstrated that the lipid kinase Vps34p is already associated with peroxisomes during biogenesis. However, although Vps34 is required, it is not essential for optimal peroxisome biogenesis. The function of Vps34p-containing complex I as well as a subset of PtdIns3P-binding proteins proved to be mandatory for the regulated degradation of peroxisomes. This demonstrates that PtdIns3P-mediated signalling is required for pexophagy.

[Anesthesia for geriatric patients : Part 2: anesthetics, patient age and anesthesia management]. / Anästhesie bei geriatrischen Patienten : Teil 2: Anästhetika, Patientenalter und Anästhesieführung.

Part 2 of this review on geriatric anesthesia primarily describes the multiple influences of age on the pharmacokinetics and pharmacodynamics of different anesthetic agents and their impact on clinical practice. In the elderly the demand for opioids is reduced by almost 50% and with total intravenous anesthesia the dosages of propofol and remifentanil as well as recovery times are more determined by patient age than by body weight. As a result depth of anesthesia monitoring is recommended for geriatric patients to individually adjust the dosing to patients needs. With muscle relaxants both delayed onset of action and prolonged duration of drug effects must be considered with increasing age and as this may lead to respiratory complications, neuromuscular monitoring is highly recommended. The following measures appear to be beneficial for geriatric patients: thorough preoperative assessment, extended hemodynamic monitoring, use of short-acting anesthetics in individually adjusted doses best tailored by depth of anesthesia monitoring, intraoperative normotension, normothermia and normocapnia, complete neuromuscular recovery at the end of the procedure and well-planned postoperative pain management in order to reduce or avoid the use of opioids.

[Anesthesia for geriatric patients. Part 1: age, organ function and typical diseases]. / Anästhesie bei geriatrischen Patienten. Teil 1: Alter, Organfunktion und typische Erkrankungen.

Due to demographic changes in the population of industrial nations the number of elderly patients undergoing elective or emergency procedures will rise significantly in the coming years. Anesthesia for geriatric patients is challenging for the anesthesiologist in many ways: with increasing age numerous physiological changes occur which all lead to a subsequent reduction of physical performance and compensatory capacity of the organism, in many cases additionally aggravated by chronic illness. Subsequently, these age-dependent changes (with or without chronic illness) increase the risk for admission to intensive care units, perioperative death, treatment costs and a prolonged length of hospital stay. Therefore, subtle preoperative assessment and tailored anesthetic management are essential in elderly patients. Part 1 of this continuous education article covers the influence of age on organ functions and describes typical comorbidities which are of high relevance for the perioperative care of geriatric patients. The special features of anesthetic agents and anesthesia management in the elderly will be presented in part 2.

Defects in myelination, paranode organization and Purkinje cell innervation in the ether lipid-deficient mouse cerebellum.

Ether lipids (ELs), particularly plasmalogens, are essential constituents of the mammalian central nervous system. The physiological role of ELs, in vivo, however is still enigmatic. In the present study, we characterized a mouse model carrying a targeted deletion of the peroxisomal dihydroxyacetonephosphate acyltransferase gene that results in the complete lack of ELs. Investigating the cerebellum of these mice, we observed: (i) defects in foliation patterning and delay in precursor granule cell migration, (ii) defects in myelination and concomitant reduction in the level of myelin basic protein, (iii) disturbances in paranode organization by extending the Caspr distribution and disrupting axo-glial septate-like junctions, (iv) impaired innervation of Purkinje cells by both parallel fibers and climbing fibers and (v) formation of axon swellings by the accumulation of inositol-tris-phosphate receptor 1 containing smooth ER-like tubuli. Functionally, conduction velocity of myelinated axons in the corpus callosum was significantly reduced. Most of these phenotypes were already apparent at P20 but still persisted in 1-year-old animals. In summary, these data show that EL deficiency results in severe developmental and lasting structural alterations at the cellular and network level of the cerebellum, and reveal an important role of ELs for proper brain function. Common molecular mechanisms that may underlie these phenotypes are discussed.

[Intravenous administration of lidocaine for perioperative analgesia. Review and recommendations for practical usage]. / Intravenös verabreichtes Lidocain zur perioperativen Schmerztherapie. Übersicht und praktische Handlungsempfehlungen.

Lidocaine is commonly used for regional anesthesia and nerve blocks. However, recent clinical studies demonstrated that intravenous perioperative administration of lidocaine can lead to better postoperative analgesia, reduced opioid consumption and improved intestinal motility. It can therefore be used as an alternative when epidural analgesia is contraindicated, not possible or not feasible. Apart from the sodium channel blocking effects relevant for regional anesthesia, lidocaine also has anti-inflammatory properties. Lidocaine can obviously inhibit the priming of resting neutrophilic granulocytes, which, simplified, may reduce the liberation of superoxide anions, a common pathway of inflammation after multiple forms of tissue trauma. At the authors' institutions intravenous lidocaine is primarily used for postoperative pain relief following abdominal surgery and is given as a bolus dose of 1.5-2.0 mg/kg body weight (BW) injected over 5 min followed by an infusion of 1.5 mg/kg BW/h intraoperatively and 1.33 mg/kg BW/h postoperatively in the recovery room or in the intensive care unit (ICU). The lidocaine infusion is stopped in the recovery room 30 min before discharge or in the ICU at the latest after 24 h. Lidocaine is not used on normal wards. This overview summarizes the current evidence for the intravenous administration of lidocaine for patients undergoing different types of surgery and gives practical advice for its use.

Assessment of Alertness and Cognitive Performance of Closed Circuit Rebreather Divers With the Critical Flicker Fusion Frequency Test in Arctic Diving Conditions.

Introduction: Cold water imposes many risks to the diver. These risks include decompression illness, physical and cognitive impairment, and hypothermia. Cognitive impairment can be estimated using a critical flicker fusion frequency (CFFF) test, but this method has only been used in a few studies conducted in an open water environment. We studied the effect of the cold and a helium-containing mixed breathing gas on the cognition of closed circuit rebreather (CCR) divers. Materials and Methods: Twenty-three divers performed an identical dive with controlled trimix gas with a CCR device in an ice-covered quarry. They assessed their thermal comfort at four time points during the dive. In addition, their skin temperature was measured at 5-min intervals throughout the dive. The divers performed the CFFF test before the dive, at target depth, and after the dive. Results: A statistically significant increase of 111.7% in CFFF values was recorded during the dive compared to the pre-dive values (p < 0.0001). The values returned to the baseline after surfacing. There was a significant drop in the divers' skin temperature of 0.48°C every 10 min during the dive (p < 0.001). The divers' subjectively assessed thermal comfort also decreased during the dive (p = 0.01). Conclusion: Our findings showed that neither extreme cold water nor helium-containing mixed breathing gas had any influence on the general CFFF profile described in the previous studies from warmer water and where divers used other breathing gases. We hypothesize that cold-water diving and helium-containing breathing gases do not in these diving conditions cause clinically relevant cerebral impairment. Therefore, we conclude that CCR diving in these conditions is safe from the perspective of alertness and cognitive performance.

Disruption of blood-testis barrier dynamics in ether-lipid-deficient mice.

One of the major roles of Sertoli cells is to establish the blood-testis (Sertoli cell) barrier (BTB), which is permanently assembled and disassembled to accommodate the translocation of leptotene spermatocytes from the basal into the adluminal compartment of the seminiferous epithelium and to guarantee completion of meiosis and spermiogenesis. Recently, we have demonstrated spermatogenesis to be arrested before spermatid elongation in Gnpat-null mice with selective deficiency of ether lipids (ELs) whose functions are poorly understood. In this study, we have focused on the spatio-temporal expression of several BTB tight-junctional proteins in the first wave of spermatogenesis to obtain insights into the physiological role of ELs during BTB establishment and dynamics. Our data confirm the transient existence of Russell's intermediate or translocation compartment delineated by two separate claudin-3-positive luminal and basal tight junctions and reveal that EL deficiency blocks BTB remodeling. This block is associated with (1) downregulation and mistargeting of claudin-3 and (2) impaired BTB disassembly resulting in deficient sealing of the intermediate compartment as shown by increased BTB permeability to biotin. These results suggest that ELs are essential for cyclic BTB dynamics ensuring the sluice mechanism for leptotene translocation into the adluminal compartment.

[Ketamine racemate and fast track anaesthesia. Influence on recovery times and postoperative opioid needs]. / Ketaminrazemat bei "Fast-track"-Anästhesie. Einfluss auf Aufwachzeiten und postoperativen Opioidbedarf.

INTRODUCTION: In this study the impact of 25 mg of ketamine racemate given just before surgery on recovery times and postoperative analgesic needs in patients undergoing vaginal hysterectomy and receiving propofol-remifentanil anaesthesia was investigated. METHODS: With ethics committee approval 70 female patients aged 25-65 years were enrolled. All patients received a total intravenous anaesthesia with remifentanil and propofol with the propofol infusion being controlled to a Narcotrend index of 40. Patients in the ketamine group (n=35) received additionally a bolus dose of 25 mg ketamine racemate intravenously 3 min before skin incision. In addition to monitoring haemodynamics and circulation parameters, recovery times, postoperative pain and opioid needs were also recorded. Patients were also questioned on their satisfaction with the pain therapy. RESULTS: All 70 patients completed the study and the groups were similar with respect to demographic data. The haemodynamics of the patients were stable in both groups and the postoperative pain measured over a 24-h period as well as the opioid needs were also comparable. However, recovery times were significantly prolonged in the ketamine group, e.g. the times to extubation were 8.3+/-4.0 min with ketamine compared to 6.1+/-2.1 min in the control group (p<0.01). Undesired side effects were overall rare but occurred to the same extent in both groups. CONCLUSIONS: This study demonstrated that 25 mg ketamine racemate given just before surgery significantly prolongs recovery times without reducing post-operative analgesic needs when applied to patients undergoing vaginal hysterectomy and receiving propofol-remifentanil anaesthesia. A bolus dose of 25 mg ketamine racemate cannot therefore be recommended for preemptive analgesia under these conditions.

[Optimization of perioperative management in laparoscopic hernioplasty]. / Optimierung des perioperativen Managements am Beispiel der laparoskopischen Leistenbruchoperation.

INTRODUCTION: Although about 150,000-200,000 hernia repair procedures are performed every year in Germany alone, fast-track concepts are mainly ignored for this type of surgery. Therefore, this study was designed to analyze the perioperative management of patients undergoing laparoscopic hernia repair, performed as transabdominal preperitoneal patch hernioplasty (TAPP). Based on these results, the clinical management was optimized in order to shorten the length of stay without affecting the quality of treatment or the complication rate, and the effects of the optimization strategies were reanalyzed. METHODS: With ethics committee approval 249 patients undergoing TAPP procedures were investigated. In the first two study sections, problems of the perioperative management were identified first retrospectively (n=129) and then prospectively (n=60). Based on these results, the clinical management was then redesigned and optimized. A TAPP score was developed including the parameters age, ASA physical status, extent and severity of the procedure and postoperative pain level. Patients were scored 24 h after surgery, and clear-cut criteria were defined for discharge home on the second postoperative day. Furthermore, all patients received 10 mg of sodium picosulfate to avoid postoperative constipation. In the third study section (n=60) the impact of the optimization strategies on length of stay, need for pain medication, complication rate and patient satisfaction (based on the PPP33 questionnaire) was evaluated. RESULTS: Patients were on average approximately 60 years old (range 22-92 years), and demographic data were comparable within the study sections. As a result of the optimization process, 72% of the patients could be discharged home on the second postoperative day whereas previously that had only been possible in 5%. Accordingly, the postoperative length of stay (including the day of surgery) was significantly reduced from 4.2+/-0.6 to 3.3+/-0.6 days. By the administration of sodium picosulfate, 92% of all patients reported defecation on the first day after surgery, whereas previously the majority of patients (60%) had complained of constipation at this time. No serious complications were observed. The number of minor complications was very low in total and neither complication rate nor patient satisfaction was affected by earlier discharge. The second day after surgery was judged to be the ideal time point for discharge by 81% of all patients. CONCLUSIONS: This study demonstrates that the length of stay after laparoscopic hernia repair can be reduced by one day by relatively simple means without affecting patient satisfaction or the complication rate. Most important is the introduction of clear-cut criteria that allow an early decision-making for discharge home. Moreover, many patients complain of constipation after laparoscopic surgery, and this may prolong the length of stay. This problem can be solved completely by the standardized use of sodium picosulfate, administered on the evening after surgery.

Peroxisome biogenesis: where Arf and coatomer might be involved.

The present review summarizes recent observations on binding of Arf and COPI coat to isolated rat liver peroxisomes. The general structural and functional features of both Arf and coatomer were considered along with the requirements and dependencies of peroxisomal Arf and coatomer recruitment. Studies on the expression of mammalian Pex11 proteins, mainly Pex11alpha and Pex11beta, intimately related to the process of peroxisome proliferation, revealed a sequence of individual steps including organelle elongation/tubulation, formation of membrane and matrix protein patches segregating distinct proteins from each other, development of membrane constrictions and final membrane fission. Based on the similarities of the processes leading to cargo selection and concentration on Golgi membranes on the one hand and to the formation of peroxisomal protein patches on the other hand, an implication of Arf and COPI in distinct processes of peroxisomal proliferation is hypothesized. Alternatively, peroxisomal Arf/COPI might facilitate the formation of COPI-coated peroxisomal vesicles functioning in cargo transport and retrieval from peroxisomes to the ER. Recent observations suggesting transport of Pex3 and Pex19 during early steps of peroxisome biogenesis from the ER to peroxisomes inevitably propose such a retrieval mechanism, provided the ER to peroxisome pathway is based on transporting vesicles.

The ether lipid-deficient mouse: tracking down plasmalogen functions.

Chemical and physico-chemical properties as well as physiological functions of major mammalian ether-linked glycerolipids, including plasmalogens were reviewed. Their chemical structures were described and their effect on membrane fluidity and membrane fusion discussed. The recent generation of mouse models with ether lipid deficiency offered the possibility to study ether lipid and particularly plasmalogen functions in vivo. Ether lipid-deficient mice revealed severe phenotypic alterations, including arrest of spermatogenesis, development of cataract and defects in central nervous system myelination. In several cell culture systems lack of plasmalogens impaired intracellular cholesterol distribution affecting plasma membrane functions and structural changes of ER and Golgi cisternae. Based on these phenotypic anomalies that were accurately described conclusions were drawn on putative functions of plasmalogens. These functions were related to cell-cell or cell-extracellular matrix interactions, formation of lipid raft microdomains and intracellular cholesterol homeostasis. There are several human disorders, such as Zellweger syndrome, rhizomelic chondrodysplasia punctata, Alzheimer's disease, Down syndrome, and Niemann-Pick type C disease that are distinguished by altered tissue plasmalogen concentrations. The role plasmalogens might play in the pathology of these disorders is discussed.

Probing the Role of Integrins in Keratinocyte Migration Using Bioengineered Extracellular Matrix Mimics.

Bioengineered extracellular matrix (ECM) mimetic materials have tunable properties and can be engineered to elicit desirable cellular responses for wound repair and tissue regeneration. By incorporating relevant cell-instructive domains, bioengineered ECM mimics can be designed to provide well-defined ECM-specific cues to influence cell motility and differentiation. More importantly, bioengineered ECM surfaces are ideal platforms for studying cell-material interactions without the need to genetically alter the cells. Here, we showed that bioengineered ECM mimics can be employed to clarify the role of integrins in keratinocyte migration. Particularly, the roles of α5ß1 and α3ß1 in keratinocytes were examined, given their known importance in keratinocyte motility. Two recombinant proteins were constructed; each protein contains a functional domain taken from fibronectin (FN-mimic) and laminin-332 (LN-mimic), designed to bind α5ß1 and α3ß1, respectively. We examined how patient-derived primary human keratinocytes migrate when sparsely seeded as well as when allowed to move collectively. We found, consistently, that FN-mimic promoted cell migration while the LN-mimic did not support cell motility. We showed that, when keratinocytes utilize α5ß1 integrins on FN-mimics, they were able to form stable focal adhesion plaques and stabilized lamellipodia. On the other hand, keratinocytes on LN-mimic utilized primarily α3ß1 integrins for migration and, strikingly, cells were unable to activate Rac1 and form stable focal adhesion plaques. Taken together, employment of our bioengineered mimics has allowed us to clarify the roles of α5ß1 and α3ß1 integrins in keratinocyte migration, as well as further provided a mechanistic explanation for their differences.

Phosphoinositide synthesis and degradation in isolated rat liver peroxisomes.

Analyzing peroxisomal phosphoinositide (PId(#)) synthesis in highly purified rat liver peroxisomes we found synthesis of phosphatidylinositol 4-phosphate (PtdIns4P), PtdIns(4,5)P(2) and PtdIns(3,5)P(2). PtdIns3P was hardly detected in vitro, however, was observed in vivo after [(32)P]-phosphate labeling of primary rat hepatocytes. In comparison with other subcellular organelles peroxisomes revealed a unique PId pattern suggesting peroxisomal specificity of the observed synthesis. Use of phosphatase inhibitors enhanced the amount of PtdIns4P. The results obtained provide evidence that isolated rat liver peroxisomes synthesize PIds and suggest the association of PId 4-kinase and PId 5-kinase and PId 4-phosphatase activities with the peroxisomal membrane.