Intermediates in membrane fusion and bilayer/nonbilayer phase transitions imaged by time-resolved cryo-transmission electron microscopy.

Bilayer-to-nonbilayer phase transitions in phospholipids occur by means of poorly characterized intermediates. Many have proposed that membrane fusion can also occur by formation of these intermediates. Structures for such intermediates were proposed in a recent theory of these transition mechanisms. Using time-resolved cryo-transmission electron Microscopy (TRC-TEM), we have directly visualized the evolution of inverted phase micro-structure in liposomal aggregates. We have identified one of the proposed intermediates, termed an interlamellar attachment (ILA), which has the structure and dimensions predicted by the theory. We show that ILAs are likely to be the structure corresponding to "lipidic particles" observed by freeze-fracture electron microscopy. ILAs appear to assemble the inverted cubic (III) phase by formation of an ILA lattice, as previously proposed. ILAs are also observed to mediate membrane fusion in the same systems, on the same time scale, and under nearly the same conditions in which membrane fusion was observed by fluorescence methods in earlier studies. These earlier studies indicated a linkage between a membrane fusion mechanism and III phase formation. Our micrographs suggest that the same intermediate structure mediates both of those processes.     

Anaerobic metabolism of Nitrosomonas europaea

Nitrosomonas europaea is capable of maintaining an anaerobic metabolism, using pyruvate as an electron donor and nitrite as an electron acceptor; utilization of nitrite depends upon supply of both pyruvate and ammonia. The role of ammonia in this reaction was not determined. N europaea also assimilates CO₂ anaerobically into cell material in the presence of nitrite (0.5–1.0 mM), pyruvate and ammonia. This reaction was partially inhibited by nitrite which apparently competed with CO₂ for reducing power. Anaerobic nitrite respiration is sensitive to ionophores, FCCP being the most effective.Non-standard-abbreviations TCA trichloroacetic acid - FCCP carbonylcyanide-p-trifluoromethoxyphenylhydrazon     

Formation of tight junctions in epithelial cells. I. Induction by proteases in a human colon carcinoma cell line

The experimental modulation of tight junctions (TJ) was studied in the human adenocarcinoma cell line HT 29 by freeze-fracture electron microscopy. The cell line has virtually no TJ when grown in culture. TJ could be induced by mild treatment with a variety of endopeptidases (trypsin, chymotrypsin, collagenase, elastase, plasmin, thrombin, papain, and pronase). Pronase induced the formation of TJ at low (but not at high) concentrations. All exopeptidases studied were unable to induce the formation of TJ. At 0 degree C the trypsin-induced formation of TJ was greatly slowed down although not entirely inhibited. However, when cells were briefly treated with trypsin at 0 degree C and subsequently transferred to 37 degrees C in the presence of protease inhibitors, TJ were rapidly assembled. Thus an induction phase at low temperature and an assembly phase at high temperature could be experimentally separated. When cells were briefly trypsinized at 0 degrees and subsequently kept at 0 degree C without trypsin for several hours, TJ still formed abundantly upon incubation at 37 degrees C. It appears therefore that the effect produced by the protease is retained for long periods in the cold.     

Hydrocortisone inhibits antigen-induced rise in intracellular free calcium concentration and abolishes leukotriene C4 production in leukemic basophils

Antigenic stimulation of rat basophilic leukemia cells (RBL-3H3) elevates intracellular free Ca²⁺ concentration ([Ca²⁺]_i) and induces production of leukotriene C₄ (LTC₄). This model was used to examine the role of Ca²⁺ in LTC₄ formation, and inhibition by hydrocortisone (HC). HC, at a physiological concentration (2×10⁻⁷M), selectively prevented the stimulatory effect of the antigen on LTC₄ production whereas the response to calcium inophore (A23187) remained unimpaired. The inhibition by HC was time-dependent: half maximal response was reached at 2 hour and maximal response at 3 hours. Addition of arachidonic acid (3 μg/ml) did not overcome the inhibitory action of HC. An elevated [Ca²⁺]_i is known to be essential for the activation ob both 5-lipoxygenase and phospholipase A₂. The stimulatory effect of the antigen on LTC₄ production was abolished when the cells were incubated in Ca²⁺-deficient medium. Likewise, calcium ionophore stimulation shows dependence on extracellular Ca²⁺. Half maximal stimulation by the antigen and calcium ionophore was observed at external Ca²⁺ concentration of 150 μM and 40 μM respectively. Treatment with HC largely prevented the antigen-induced rise in [Ca²⁺]_i, measured by Quin 2. In addition, HC reduced by 70% the accumulation of ⁴⁵Ca²⁺ induced by the antigen. Collectively, these results demonstrate for the first time that HC reduces antigen-induced elevation of [Ca²⁺]_i, and this may be associated with the inhibitory action of HC on LTC₄ formation. This property could be partly responsible for the antiallergic and antiinflammatory activities of HC.     

Inter-Individual Differences in Neurobehavioural Impairment following Sleep Restriction Are Associated with Circadian Rhythm Phase

Although sleep restriction is associated with decrements in daytime alertness and neurobehavioural performance, there are considerable inter-individual differences in the degree of impairment. This study examined the effects of short-term sleep restriction on neurobehavioural performance and sleepiness, and the associations between individual differences in impairments and circadian rhythm phase. Healthy adults (n = 43; 22 M) aged 22.5 ± 3.1 (mean ± SD) years maintained a regular 8:16 h sleep:wake routine for at least three weeks prior to laboratory admission. Sleep opportunity was restricted to 5 hours time-in-bed at home the night before admission and 3 hours time-in-bed in the laboratory, aligned by wake time. Hourly saliva samples were collected from 5.5 h before until 5 h after the pre-laboratory scheduled bedtime to assess dim light melatonin onset (DLMO) as a marker of circadian phase. Participants completed a 10-min auditory Psychomotor Vigilance Task (PVT), the Karolinska Sleepiness Scale (KSS) and had slow eye movements (SEM) measured by electrooculography two hours after waking. We observed substantial inter-individual variability in neurobehavioural performance, particularly in the number of PVT lapses. Increased PVT lapses (r = -0.468, p < 0.01), greater sleepiness (r = 0.510, p < 0.0001), and more slow eye movements (r = 0.375, p = 0.022) were significantly associated with later DLMO, consistent with participants waking at an earlier circadian phase. When the difference between DLMO and sleep onset was less than 2 hours, individuals were significantly more likely to have at least three attentional lapses the following morning. This study demonstrates that the phase of an individual’s circadian system is an important variable in predicting the degree of neurobehavioural performance impairment in the hours after waking following sleep restriction, and confirms that other factors influencing performance decrements require further investigation.     

Remote monitoring and follow-up of cardiovascular implantable electronic devices in the Netherlands : An expert consensus report of the Netherlands Society of Cardiology

Remote monitoring of cardiac implanted electronic devices (CIED: pacemaker, cardiac resynchronisation therapy device and implantable cardioverter defibrillator) has been developed for technical control and follow-up using transtelephonic data transmission. In addition, automatic or patient-triggered alerts are sent to the cardiologist or allied professional who can respond if necessary with various interventions. The advantage of remote monitoring appears obvious in impending CIED failures and suspected symptoms but is less likely in routine follow-up of CIED. For this follow-up the indications, quality of care, cost-effectiveneness and patient satisfaction have to be determined before remote CIED monitoring can be applied in daily practice. Nevertheless remote CIED monitoring is expanding rapidly in the Netherlands without professional agreements about methodology, responsibilities of all the parties involved and that of the device patient, and reimbursement. The purpose of this consensus document on remote CIED monitoring and follow-up is to lay the base for a nationwide, uniform implementation in the Netherlands. This report describes the technical communication, current indications, benefits and limitations of remote CIED monitoring and follow-up, the role of the patient and device manufacturer, and costs and reimbursement. The view of cardiology experts and of other disciplines in conjunction with literature was incorporated in a preliminary series of recommendations. In addition, an overview of the questions related to remote CIED monitoring that need to be answered is given. This consensus document can be used for future guidelines for the Dutch profession.     

H5-type influenza virus hemagglutinin is functionally recognized by the natural killer-activating receptor NKp44

Antiviral immune defenses involve natural killer (NK) cells. We previously showed that the NK-activating receptor NKp44 is involved in the functional recognition of H1-type influenza virus strains by NK cells. In the present study, we investigated the interaction of NKp44 and the hemagglutinin of a primary influenza virus H5N1 isolate. Here we show that recombinant NKp44 recognizes H5-expressing cells and specifically interacts with soluble H5 hemagglutinin. H5-pseudotyped lentiviral particles bind to NK cells expressing NKp44. Following interaction with target cells expressing H5, pseudotyped lentiviral particles, or membrane-associated H5, NK cells show NKp44-mediated induced activity. These findings indicate that NKp44-H5 interactions induce functional NK activation.     

Germination Studies ofLesquerella fendleri andL. gordonii,with reference to their cultivation

Seeds ofLesqurella spices are known to be dormant for five years and more. In oder to make cultivation ofL. fendleri andL. gordonii feasible germination studies were undertaken and practical applications of the results were investigated.