bluuebunny/crossref_metadata_2025 huggingface dataset:
Tracking of secretory vesicles of PC12 cells by total internal reflection fluorescence microscopy
Authors: Yang D.‐M. , Huang C.‐C. , Lin H.‐Y. , Tsai D.‐P. , Kao L.‐S. , Chi C.‐W. , Lin C.‐C.
Published: October 2010

Total internal reflection fluorescence microscopy is used to detect cellular events near the plasma membrane. Behaviours of secretory vesicles near the cell surface of living PC12 cells, a neuroendocrine cell line, are studied. The secretory vesicles are labelled by over‐expression of enhanced green fluorescent protein‐tagged Rab3A, one of the small G proteins involved in the fusion of secretory vesicles to plasma membrane in PC12 cells. Images acquired by a fast cooled charge‐coupled device camera using conventional fluorescence microscopy and total internal reflection fluorescence microscopy are compared and analysed. Within the small evanescent range (< 200 nm), the movements of the secretory vesicles of PC12 cells before and after stimulation by high K+ are examined. The movements of one vesicle relative to another already docked on the membrane are detected. Total internal reflection fluorescence microscopy provides a novel optical method to trace and analyse the exocytotic events and vesicle specifically near a cell membrane without interference of signals from other parts of the cell.
Near‐field imaging of surface plasmon on gold nano‐dots fabricated by scanning probe lithography
Authors: Kim J. , Kim J. , Song K. I.‐B. , Lee S. Q. , Kim E. U. N.‐K. , Choi S.‐E. U. L. , Lee Y. , Park K.‐H. O.
Published: October 2010

We obtained scanning near‐field optical microscopy images to study the excitation of surface plasmons on metallic dots fabricated using scanning probe lithography. Gold nano‐dots were fabricated by applying electric voltages to conducting probes installed in an atomic force microscope using the mechanism of field‐induced diffusion and nano‐oxidation plus Au‐coating. High spatial resolution of scanning near‐field optical microscopy revealed a ‘bifold’ pattern of surface plasmon mode on fabricated Au dots in the polarization direction of incident light. We found that scanning near‐field optical microscopy imaging combined with scanning probe lithography is able to provide a systematic study of surface plasmon excitation on nano‐metallic structures.
Photoplastic near‐field optical probe with sub‐100 nm aperture made by replication from a nanomould
Authors: Kim G. M. , Kim B. J. , Ten Have E. S. , Segerink F. , Van Hulst N. F. , Brugger J.
Published: October 2010

Polymers have the ability to conform to surface contours down to a few nanometres. We studied the filling of transparent epoxy‐type EPON SU‐8 into nanoscale apertures made in a thin metal film as a new method for polymer/metal near‐field optical structures. Mould replica processes combining silicon micromachining with the photo‐curable SU‐8 offer great potential for low‐cost nanostructure fabrication. In addition to offering a route for mass production, the transparent pyramidal probes are expected to improve light transmission thanks to a wider geometry near the aperture. By combining silicon MEMS, mould geometry tuning by oxidation, anti‐adhesion coating by self‐assembled monolayer and mechanical release steps, we propose an advanced method for near‐field optical probe fabrication. The major improvement is the possibility to fabricate nanoscale apertures directly on wafer scale during the microfabrication process and not on free‐standing tips. Optical measurements were performed with the fabricated probes. The full width half maximum after a Gaussian fit of the intensity profile indicates a lateral optical resolution of ≈ 60 nm.
Implementation of a short‐tip tapping‐mode tuning fork near‐field scanning optical microscope
Authors: Lu N. H. , Huang C. W. , Chen C. Y. , Yu C. F. , Kao T. S. , Fu Y. H. , Tsai D. P.
Published: October 2010

We present the implementation of a short‐tip tapping‐mode tuning fork near‐field scanning optical microscope. Tapping frequency dependences of the piezoelectric signal amplitudes for a bare tuning fork fixed on the ceramic plate, a short‐tip tapping‐mode tuning fork scheme and an ordinary tapping‐mode tuning fork configuration with an 80‐cm optical fibre attached are demonstrated and compared. Our experimental results show that this new short‐tip tapping‐mode tuning fork scheme provides a stable and high Q factor at the tapping frequency of the tuning fork and will be very helpful when long optical fibre probes have to be used in an experiment. Both collection and excitation modes of short‐tip tapping‐mode tuning fork near‐field scanning optical microscope are applied to study the near‐field optical properties of a single‐mode telecommunication optical fibre and a green InGaN/GaN multiquantum well light‐emitting diode.
High‐resolution backscatter electron imaging of colloidal gold in LVSEM
Authors: Erlandsen S. , Chen Y. , Frethem C. , Detry J. , Wells C.
Published: October 2010

High‐resolution backscatter electron (BSE) imaging of colloidal gold can be accomplished at low voltage using in‐lens or below‐the‐lens FESEMs equipped with either Autrata‐modified yttrium aluminium garnet (YAG) scintillators doped with cerium, or with BSE to secondary electron (SE) conversion plates. The threshold for BSE detection of colloidal gold was 1.8 keV for the YAG detector, and the BSE/SE conversion was sensitive down to 1 keV. Gold particles (6, 12 and 18 nm) have an atomic number of 79 and were clearly distinguished at 500 000× by materials contrast and easily discriminated from cell surfaces coated with platinum with an atomic number of 78. BSE imaging was relatively insensitive to charging, and build up of carbon contamination on the specimen was transparent to the higher energy BSE.
A comparative study between AFM and SEM imaging on human scalp hair
Authors: Poletti G. , Orsini F. , Lenardi C. , Barborini E.
Published: October 2010

A comparative study of AFM and SEM imaging of the same area of a human scalp hair has been carried out to determine the similarity and the differences between the two techniques. Sample preparation for SEM analysis requires a metallization step and vacuum exposure, both of which could potentially induce modifications to the surface details. By contrast, AFM is a suitable technique to evaluate any effect resulting from sample manipulation because it can be applied without any specific treatment. AFM analysis demonstrates that sample metallization is responsible for modifications to the surface details of hair, mainly comprising an increase in height of scale steps and of root mean square roughness together with variation in scale profiles. Sample treatments for SEM imaging are in general potentially responsible for surface modifications to the samples involved.
Decreased uptake of bodipy‐labelled compounds in the presence of the nuclear stain, DRAQ5
Authors: Snyder D. Scott , Garon C. F.
Published: October 2010

We have found the nuclear stain DRAQ5 to decrease the cellular uptake of a series of boron dipyrromethane (bodipy)‐labelled compounds. This phenomenon is consistent between Lysotracker Green DND 26, Lysotracker Red DND 99 and bodipy‐labelled mycolactone. Although DRAQ5 uptake was not prevented, DRAQ5 was in significant excess in each case. As the effect is consistent among two cell types, RAW 264.7 monocyte/macrophages and Bend 3 endothelial cells, we hypothesize that it may be a result of the two dyes complexing in solution into a form that is not taken up by the cells. This hypothesis is confirmed by fluorescence resonance energy transfer analysis in solution.
Quantitative fluorescent speckle microscopy: where it came from and where it is going
Authors: Danuser G. , Waterman‐Storer C. M.
Published: October 2010

Fluorescent speckle microscopy (FSM) is a technology for analysing the dynamics of macromolecular assemblies. Originally, the effect of random speckle formation was discovered with microtubules. Since then, the method has been expanded to other proteins of the cytoskeleton such as f‐actin and microtubule binding proteins. Newly developed, specialized software for analysing speckle movement and photometric fluctuation in the context of polymer transport and turnover has turned FSM into a powerful method for the study of cytoskeletal dynamics in cell migration, division, morphogenesis and neuronal path finding. In all these settings, FSM serves as the quantitative readout to link molecular and genetic interventions to complete maps of the cytoskeleton dynamics and thus can be used for the systematic deciphering of molecular regulation of the cytoskeleton. Fully automated FSM assays can also be applied to live‐cell screens for toxins, chemicals, drugs and genes that affect cytoskeletal dynamics. We envision that FSM has the potential to become a core tool in automated, cell‐based molecular diagnostics in cases where variations in cytoskeletal dynamics are a sensitive signal for the state of a disease, or the activity of a molecular perturbant. In this paper, we review the origins of FSM, discuss these most recent technical developments and give a glimpse to future directions and potentials of FSM. It is written as a complement to the recent review (Waterman‐Storer & Danuser, 2002, Curr. Biol., 12, R633–R640), in which we emphasized the use of FSM in cell biological applications. Here, we focus on the technical aspects of making FSM a quantitative method.
Automatic fluorescent tag localization II: improvement in super‐resolution by relative tracking
Authors: Thomann D. , Dorn J. , Sorger P. K. , Danuser G.
Published: October 2010

We present an algorithm for the three‐dimensional (3D) tracking of multiple fluorescent subresolution tags with super‐resolution in images of living cells. Recently, we described an algorithm for the automatic detection of such tags in single frames and demonstrated its potential in a biological system. The algorithm presented here adds to the tag detector a module for relative tracking of the signals between frames. As with tag detection, the main problem in relative tracking arises when signals of multiple tags interfere. We propose a novel multitemplate matching framework that exploits knowledge of the microscope point spread function to separate the intensity contribution of each tag in image regions with signal interferences. We use this intensity splitting to reconstruct a template for each tag in the source frame and a patch in the target frame, which are both free of intensity contributions from other tag signals. Tag movements between frames are then tracked by seeking, for each template–patch pair, the displacement vector providing the best signal match in terms of the sum of squared intensity differences. Because template and patch generation of tags with overlapping signals are interdependent, the matching is carried out simultaneously for all tags, and in an iterative manner. We have examined the performance of our approach using synthetic 3D data and observed a significant increase in resolution and robustness as compared with our previously described detector. It is now possible to localize and track tags separated by a distance three times smaller than the Rayleigh limit with a relative positional accuracy of better than 50 nm. We have applied the new tracking system to extract metaphase trajectories of fluorescently tagged chromosomes relative to the spindle poles in budding yeast.
A review of research on the structure, process and outcome of liaison mental health services
Authors: CALLAGHAN P. , EALES S. , COATES T. , BOWERS L.
Published: October 2010

Liaison mental health services (LMHS) developed originally to address the mental health needs of people with physical illnesses in general hospitals and more recently to work also with people with mental health problems presenting at non‐mental health services. The purpose of the present paper was to review empirical research on the structure, process and outcome of liaison mental health services using systematic review methods. Following a comprehensive search strategy, the authors reviewed 48 papers published between 1975 and 2001. There is an extensive international literature on LMHS, much of which describes the structure and process of liaison work. Studies evaluating the outcomes of liaison mental health services are fewer, and handicapped by methodological flaws, some of which are serious enough to cast doubts on the reported results. Professionals and clients value LMHS. LMHS based in accident and emergency (A & E) departments appear to ease the burden of general A & E staff, help clients access mental health services and reduce re‐admission rates of people with mental health problems. There is little evidence supporting one model of configuring LMHS over another.
People with dementia and their relatives: personal experiences of Alzheimer's and of the provision of care
Authors: AGGARWAL N. , VASS A. A. , MINARDI H. A. , WARD R. , GARFIELD C. , CYBYK B.
Published: October 2010

Little is known about the experiences of people with dementia, as there has been relatively little research carried out that includes their perspectives. In the past, user perspectives on experiences of dementia and on services have mostly been solicited from family carers, rather than from people with dementia themselves. It has been suggested that these studies may lack information and insight into the experiences of people with dementia. This study aimed at eliciting the views and feelings of people in all stages of dementia, as well as those of their relatives, on care services and on experiences of dementia. Twenty‐seven people with dementia from residential and day care settings were interviewed and their daily lives videotaped. Interviews were also conducted with next of kin. This article reports on findings and issues arising from the study.
Person first: what people with enduring mental disorders value about community psychiatric nurses and CPN services
Authors: ADAM R. , TILLEY S. , POLLOCK L.
Published: October 2010

Based on a Scottish study, this article presents findings from qualitative analysis of interview data, on views of people with enduring mental disorders (people) regarding services provided by community psychiatric nurses (CPNs) and what these people value in working with CPNs. Thirteen people took part in semi‐structured interviews, and data were analysed using strategies including thematic analysis. The main finding was that people value their interpersonal relationship with CPNs. This relationship has a specific function in the individual's overall social network. The CPN–person relationship forms the context of ‘purposeful talk’, and is shaped and developed through the talk. It provides comfort and a greater sense of confidence with which people can cope with daily life. A valued feature of the personalized relationship with CPNs is continuity, associated with regularity of contact with CPNs, accessibility (both physical and emotional) and respect for and commitment to people as individuals. We interpret CPNs, as they appear in these accounts given by people with enduring mental disorders, as ‘beings‐in‐between’, bridging symbolically the worlds of hospital and community. They are figures between ‘friends’ and professionals, to whom people who have been ill can relate and show feelings which would, if otherwise expressed, compromise participation in community. CPNs help sustain people experiencing ‘illness in the context of life’ and enhance their potential for participation in the community. In doing so they contribute to public health. Viability of sustaining relationships and personal care, valued at the micro‐level of interaction, depends on support at the meso‐level by managers, and at the macro‐level by policy makers and funders. Health service managers who play a key role in instigating and managing service changes should engage in regular dialogue with CPNs about the impact of change on the ability of CPNs to maintain sustaining relationships with people. The adaptability of CPNs to the situation and the person‐in‐the‐situation needs to be facilitated, not compromised, by the requirements of record‐keeping and accounting systems. Practice described in this study indicates the possibility of CPNs relating to the person in ways consistent with a ‘community development approach’. Limitations of the study are noted. Our findings are highly contextualized and based on a small sample. Nonetheless, they are consistent with findings from other studies also based on listening to service users’ accounts of problems of living with enduring mental disorder. Implications of the analysis of peoples’ perceptions of the role of CPNs are considered, with attention to service providers, policy makers and future research.
Patients’ satisfaction with outpatient psychiatric care
Authors: SIPONEN U. , VÄLIMÄKI M.
Published: October 2010

The purpose of this study was to describe patient satisfaction with outpatient psychiatric care in two community care clinics in Finland. Data were collected using a structured questionnaire from 300 outpatients between February and March 2000. A total of 171 patients returned completed questionnaires (response rate 57%). The data analysis was based on descriptive statistics, chi‐square test, the Mann–Whitney U‐test and the Kruskal–Wallis test. Item homogeneity was analysed using Cronbach alpha coefficient. The results showed that patient satisfaction was highest in areas pertaining to staff and care discussions, and lowest in areas pertaining to information. Patients who considered the wait for treatment to be too long were more dissatisfied with staff, their chance of influencing their own care, and the help they received, compared with patients who considered the wait to be reasonable in length. We can conclude that although patients are quite satisfied with their outpatient treatment in general, the patient information process is not at a satisfactory level at outpatient psychiatric clinics. In the future, more emphasis should be put on developing more innovative methods to increase psychiatric patients’ knowledge level regarding their own illness and treatment.
Patients’ experiences of psychosis in an inpatient setting
Authors: KOIVISTO K. , JANHONEN S. , VAISANEN L.
Published: October 2010

The aim of this report was to describe patients’ experiences of psychosis in an inpatient setting. Mental illness, as a result of psychosis, has traditionally been defined from the viewpoint of clinical experts. Psychiatric nursing, as an interactive human activity, is more concerned with the development of the person than with the origins or causes of their present distress. Therefore, psychiatric nursing is based on eliciting personal experiences and assisting the person to reclaim her/his inner wisdom and power. The design of the study, in the report discussed below, was phenomenological. In 1998, nine patients were interviewed regarding their experiences of psychosis in an acute inpatient setting. The verbatim transcripts were analysed using Giorgi's phenomenological method. The participants experienced psychosis as an uncontrollable sense of self, which included feelings of change and a loss of control over one's self with emotional distress and physical pain. The participants described the vulnerability they had felt whilst having difficult and strange psychological feelings. The informants experienced both themselves and others sensitively, considered their family and friends important and meaningful, and found it difficult to manage their daily lives. Furthermore, the informants experienced the onset of illness as situational, the progress of illness as holistic and exhaustive, and the admission into treatment as difficult, but inevitable.
Diurnal changes of blood pressure, heart rate and body temperature during sleep in the rat
Authors: SEI HIROYOSHI , FURUNO NARIO , MORITA YUSUKE
Published: October 2010

We have studied diurnal changes in mean arterial pressure (MAP), heart rate (HR) and body temperature (Tb) during wake (W), non‐rapid eye movement sleep (NREMS) and REM sleep (REMS) in the rat. Although HR and Tb show a similar sinusoidal diurnal variation during all vigilance states, the diurnal profile for the MAP is vigilance‐state dependent. During W, MAP values are higher during the dark phase, during NREMS, no significant diurnal change is seen, and during REMS, the MAP exhibits a reversed diurnal change, being higher during the light phase. The low frequency component (0.25–0.725 Hz) in the power spectral density of the blood pressure, an index of sympathetic activity, is also higher during the light phase than the dark phase in REMS. The present findings suggest that diurnal changes in MAP in the rat result from the wake rhythm, and that the mechanism for the diurnal control of MAP may be different from that for HR or Tb.
A behavioural test to assess daytime sleepiness in obstructive sleep apnoea
Authors: BENNETT L , STRADLING J , DAVIES R
Published: October 2010

Daytime sleepiness is an important symptom of obstructive sleep apnoea syndrome (OSAS). The standard tests for its objective quantification use EEG recordings, and are time consuming and expensive, which makes them difficult to use for large studies. This study assesses the ability of a simple test of sustained ‘wakefulness’ to discriminate the excessive somnolence of severe symptomatic obstructive sleep apnoea from normality, and compares its results to the traditional EEG based Maintenance of Wakefulness Test (MWT). Ten subjects (7M 3F) with severe sleep apnoea (>4% SaO2 dip rate median 32.7 (90% central range 9.7–65.6)) and symptoms of daytime sleepiness, (Epworth Sleepiness Score (ESS)17(10–24)) and 10 normal subjects (4M 6F, ESS 3.5(1–8)) were studied. The MWT and the behavioural test (Oxford SLEep Resistance test – OSLER test) were performed on each subject in random order on 2 separate days. The protocol for both tests was the same with 4 × 40 min sleep resistance challenges throughout the day while sound isolated in a darkened room. During the OSLER test subjects were asked to press a switch in response to a light emitting diode (LED), which was lit for 1 s in every three. Both the switch and the light were connected to a computer that stored both the number of times the light was illuminated and whether a correct response was made. The OSLER test discriminated the normal subjects from the sleep apnoea group (mean sleep latency (min) normal group 39.8, OSA group 10.5) as well as the traditional MWT (normal group 38.1, OSA group 7.3) and was much simpler to administer. This test has the advantage that sleep onset is defined objectively and automatically as a failure to respond to the light, rather than from EEG interpretation, which is inevitably partly subjective. This technique may provide a simple and robust method of objectively quantifying daytime sleepiness for large studies.
Circadian distribution of rest/activity in narcoleptic and control dogs: assessment with ambulatory activity monitoring
Authors: NISHINO S , TAFTI M , SAMPATHKUMARAN R , DEMENT W , MIGNOT E
Published: October 2010

Like human narcoleptics, narcoleptic dogs display cataplexy, fragmented sleep and excessive daytime sleepiness. Cataplexy in dogs can easily be quantified using a simple behavioural bioassay, the Food Elicited Cataplexy Test. In contrast, daytime sleepiness and fragmented sleep are more difficult to measure, as long‐term, labour‐intensive polygraphic recordings in surgically‐implanted animals are needed. In the current study, 24‐h rest/activity patterns in genetically narcoleptic, asymptomatic heterozygous and control Dobermans were compared using small sized ambulatory activity monitoring devices under 12‐h light/dark conditions. Control and heterozygous dogs were found to be more active during the light period than during the dark period, thus demonstrating a clear 24‐h rest/activity cycle. In contrast, narcoleptic dogs were relatively inactive during the light period and did not show a clear rest/activity pattern, a result similar to that of human narcoleptics. Considering the fact that narcoleptic dogs show shorter sleep latency and sleep significantly more during the daytime than control dogs, the decrease in activity in narcoleptic dogs during the daytime is most likely a reflection of increased daytime napping in these animals. Ambulatory activity monitoring may be a useful non‐invasive method for future pharmacological and developmental studies in the narcoleptic canine model.
Fronto‐occipital EEG power gradients in human sleep
Authors: WERTH ESTHER , ACHERMANN PETER , BORBÉLY ALEXANDER
Published: October 2010

The brain topography of power spectra along the antero‐posterior (A‐P) axis was studied in the all‐night human sleep EEG. Spectra (0.25–25.0 Hz) were computed for an anterior (A; F3‐C3), a middle (M; C3‐P3) and a posterior (P; P3‐O1) bipolar derivation, and the spectral gradients between two adjacent derivations were expressed by power ratios (A/M and M/P). At NREM‐REM sleep transitions a power shift from A to M was present over almost the entire frequency range, while the direction of shifts between M and P differed between frequency bands. Within NREM sleep, frequency specific power gradients were present: In the low delta band power in both A (0.25 Hz bin) and P (0.25–1.0 Hz bins) was higher than in M. In the 4–9 Hz range the relation was A>M>P, and in the 15–25 Hz range power was largest in M. Power in the spindle frequency range was highest at 11.75 Hz in M, and at 13.5–13.75 Hz in A. Topographical differences were seen also in the temporal changes of power across and within NREM sleep episodes. Whereas NREM sleep power in the 2‐Hz bin was higher in A than in M in the first episode, this difference vanished in the course of the night. This result points to a specific involvement of frontal parts of the cortex in sleep homeostasis. The regional differences in sleep EEG spectra indicate that sleep is not only a global phenomenon but also a local brain process with a different regional involvement of neuronal populations.
Obstructive sleep apnoea and upper airway reactivity
Authors: NANDWANI N , CARANZA R , HANNING C. D
Published: October 2010

Upper airway reactivity was measured in 13 patients with obstructive sleep apnoea (OSA), using transient reflex laryngeal closure in response to dilute inhaled ammonia vapour. Upper airway reactivity was measured before and after 3 months of treatment with nasal continuous positive airway pressure (CPAP). Upper airway reactivity decreased significantly after treatment with nasal CPAP to values which were similar to those seen in normal subjects. We hypothesise that patients with OSA have increased upper airway reactivity, secondary to inflammation of the epithelial lining of the upper airway following the repeated injury of nocturnal airway obstruction, allowing the facilitated passage of inhaled irritants to the subepithelial receptors. Treatment of OSA with nasal CPAP may reverse these changes, although in the absence of a control group, these findings are provisional.
Expansins in growing tomato leaves
Authors: Keller Elvira , Cosgrove Daniel J.
Published: October 2010

An expansin‐like protein from growing tomato leaves was identifed by its ability to restore the ‘acid‐growth’ response to heat‐inactivated tomato walls and by its similarity to expansins from cucumber hypocotyls. Native walls from growing tomato leaves exhibit an endogenous acid‐induced extension (creep) that resembles in various biochemical characteristics the acid‐growth activity of cucumber hypocotyls. For example, the acid‐growth activity is lost when the walls of tomato leaves are briefly heated and is largely restored by addition of a crude protein extract from the walls of growing leaves. Wall proteins from growing leaves enhance the stress relaxation spectrum of tomato walls in a fashion characteristic of cucumber expansins. HPLC fractionation of the crude wall protein from tomato leaves yielded an active fraction containing a major 27 kDa protein that cross‐reacts with an antibody raised against cucumber expansin. The results show that tomato leaf walls possess at least one expansin that is responsible for the acid‐growth property of leaves and indicate that cell wall extension in leaves shares an underlying protein mechanism common to cell wall expansion in stems.
The S locus receptor kinase gene encodes a soluble glycoprotein corresponding to the SRK extracellular domain in Brassica oleracea
Authors: Giranton Jean‐Loïc , Ariza María José , Dumas Christian , Cock J. Mark , Gaude Thierry
Published: October 2010

Self‐incompatibility in Brassica is controlled by the S locus which contains at least two genes. SLG encodes a secreted S locus glycoprotein whilst SRK encodes a putative S locus receptor kinase which consists of three domains: an extracellular domain sharing extensive sequence identity with SLG, a transmembrane region, and a cytoplasmic domain exhibiting a serine/threonine protein kinase activity. Here, the existence of truncated forms of the SRK protein corresponding to the extracellular domain of the putative receptor is reported. These proteins were detected by an antibody which recognizes the N‐terminus of SRK3 and, in an F2 progeny segregating for the S3 haplotype, were only expressed in plants possessing the S3 haplotype. The truncated SRK proteins were expressed specifically in stigmas but, unlike the membrane‐spanning SRK3 protein, were soluble and occurred as four different glycoforms sharing the same amino acid backbone as shown by deglycosylation experiments. Several SRK3 transcripts that may code for these truncated SRK3 proteins have been identified by RACE PCR, stigma cDNA library screening and RNA blot analysis. These transcripts are apparently generated by a combination of alternative splicing and the use of alternative polyadenylation signals. The existence of truncated forms of the S locus receptor kinase highlights some similarities between plant and animal receptor kinases. In animals, soluble extracellular domains of receptors have been described and, in some cases, have been shown to play a role in the modulation of signal transduction. By analogy, the soluble, truncated SRK proteins may play a similar role in the self‐incompatibility response.
A microtubule‐associated protein in maize is expressed during phytochrome‐induced cell elongation
Authors: Nick Peter , Lambert Anne‐Marie , Vantard Marylin
Published: October 2010

Plants can adapt their shape to environmental stimuli. This response is mediated by the reorganization of cortical microtubules, a unique element of the cytoskeleton. However, the molecular base of this response has remained obscure so far. In an attempt to solve this problem, signal‐dependent changes in the pattern of microtubule‐binding proteins were analysed during coleoptile elongation in maize, that is, under the control of the plant photoreceptor phytochrome. Two putative MAPs of 100 kDa (P100) and 50 kDa apparent molecular weights were identified in cytosolic extracts from non‐elongating and elongating cells. Both proteins co‐assembled with endogenous tubulin, bound to neurotubules and were immunologically related to the neural MAP τ: the P100 protein, depending on the physiological situation, was manifest as a double band and was always found to be heat‐stable. In contrast, the 50 kDa MAP was heat‐stable only for particular tissues and physiological treatments. The P100 protein was present in all tissues, however in a reduced amount in elongating coleoptiles. The 50 kDa MAP was expressed exclusively upon induction of phytochrome‐dependent cell elongation. As shown by immunofluorescence double‐staining, an epitope shared by both proteins colocalized with cortical microtubules in situ, but exclusively in elongating cells. In non‐elongating cells, only the nuclei were stained. Partially purified nuclei from elongating cells were enriched in P100, whereas the 50 kDa MAP became enriched in a partially purified plasma membrane fraction.
A repetitive DNA fragment carrying a hot spot for de novo DNA methylation enhances expression variegation in tobacco and petunia
Authors: Lohuis Michael ten , Müller Andreas , Heidmann Iris , Niedenhof Ingrid , Meyer Peter
Published: October 2010

A 1.6 kb repetitive DNA sequence (RPS) from Petunia hybrida was identified that destabilizes expression of a GUS marker transgene. Following polyethylene glycol (PEG)‐mediated tobacco and petunia protoplast transformations, GUS expression patterns analysed on callus and plant levels were clearly more variable when constructs contained the RPS sequence. The effect on transgens expression required chromosomal integration since the two different RPS constructs employed did not exhibit reduced levels of GUS activities in transient assays. DNA methylation analysis implies a hypermethylated default state of endogenous RPS copies present in the petunia genome. Analysis of the transgens DNA in different transgenic tobacco plants showed almost complete hypermethylation of a particular Hhal site of the RPS sequence. It is proposed that, due to the presence of specific signals within the RPS region or based on interaction of RPS with other endogenous homologous sequences, RPS functions as an initiation region for de novo methylation and induces expression variegation in adjacent sequences.
Characterization of the targeted nuclear accumulation of GFP within the cells of transgenic plants
Authors: Grebenok Robert , Lambert Georgina , Galbraith David
Published: October 2010

The soluble proteins of the nucleoplasm are synthesized on cytoplasmic ribosomes. Proteins larger than about 40 kDa are post‐translationally targeted to the nucleus via energy‐dependent processes, passing through the nuclear pore complex into the nucleoplasm. Targeting involves nuclear localization signals (NLSs) found within the primary sequences of the imported proteins. In higher plants, information has come primarily from study of proteins carrying ‘classical’ NLSs, comprising stretches of basic amino acids, and has required assays to measure nuclear uptake both in vitro and in vivo. In general, these assays are not entirely satisfactory; they are either technically demanding, are of limited accuracy and statistical rigor, or are unsuitable for in vivo applications.
Light‐dependent regulation of carotenoid biosynthesis occurs at the level of phytoene synthase expression and is mediated by phytochrome in Sinapis alba and Arabidopsis thaliana seedlings
Authors: Von Lintig Johannes , Welsch Ralf , Bonk Michael , Giuliano Giovanni , Batschauer Alfred , Kleinig Hans
Published: October 2010

In chloroplasts, carotenoids are essential pigments involved in photosynthesis. During photomorphogenesis, a coordinated increase in the amounts of chlorophylls and carotenoids, in conjugation with other components, leads to the formation of a functional photosynthetic apparatus. To investigate the regulation of carotenoid biosynthesis during this process at the molecular level, GGPS, PSY and PDS cDNAs have been cloned from white mustard (Sinapis alba L.). GGPS encodes a key enzyme in plastid isoprenoid metabolism, while the products of PSY and PDS catalyse the subsequent steps in carotenoid biosynthesis. Due to the low mRNA levels of the genes involved, the use of a RT–PCR protocol was necessary to measure gene expression during photomorphogenesis. With light, there is an up‐regulation of PSY expression, the first gene within the carotenoid biosynthetic pathway, while PDS and GGPS expression levels remain constant. Treatment with different light qualities reveals a phytochrome‐mediated regulation of PSY expression in developing white mustard seedlings. To obtain more detailed information on the light‐regulation, Arabidopsis thaliana wild‐type and phytochrome mutants were utilized. Continuous far‐red and red light both increase the expression of PSY in wild‐type seedlings, demonstrating that both light‐labile and light‐stable phytochromes are involved in PSY regulation. The response to far‐red light is completely abolished in the phyA mutant, showing that PHYA mediates the increase in PSY transcript levels under these light conditions. In the phyB mutant, the red light response is normal, indicating that PSY expression is not controlled by PHYB but by other light‐stable phytochromes. Measurement of chlorophylls and carotenoids under the same light regimes shows that the up‐regulation of PSY expression does not necessarily result in an increase of the carotenoid content. Only those light conditions which allow chlorophyll biosynthesis lead to a significant increase of the carotenoid content. Therefore, it is proposed that up‐regulation of PSY mRNA levels leads to an increased capacity for the formation of carotenoids. However, this only takes place under light conditions leading to protochlorophyllide photoconversion.
Cloning and characterization of LRG5, a gene involved in blue light signaling in Chlamydomonas gametogenesis
Authors: Glöckner Gernot , Beck Christoph
Published: October 2010

The Chlamydomonas reinhardtii mutant lrg2 exhibits a partial defect in the blue‐light activated signal transduction chain that controls gametogenesis. By genomic complementation, a cosmid clone that corrects the lrg2 phenotype was isolated. The smallest fragment of this cosmid that gave complementation was used in RFLP analysis, which revealed that the cloned gene was not linked to the LRG2 locus. The sequence of the genomic clone and a cDNA of this gene, which we named LRG5, was determined. Although no significant homology with sequences in the databases was found, the nuclear localization signal and the presence of negatively and positively charged domains suggests a function for the LRG5 protein in the regulation of transcription. The correction of the lrg2 phenotype is probably caused by overexpression of the LRG5 gene in the transformants, as these expressed a level of LRG5 approximately twofold higher than wild‐type or mutant strains. Southern blot analysis provided evidence for the existence of homologous sequences in other green algae as well as in higher plants, suggesting a conserved function for the LRG5 protein.
Construction of an ∼2 Mb contig in the region around 80 cM of Arabidopsis thaliana chromosome 2
Authors: Wang Ming Li , Huang Lu , Bongard‐Pierce Deverie , Belmonte Stephen , Zachgo Eve Ann , Morris John , Dolan Maureen , Goodman Howard
Published: October 2010

A method for construction of bacterial artificial chromosome (BAC) contigs from a yeast artifical chromosome (YAC) physical map is described. An ∼2 Mb contig, consisting of two large BAC contigs linked by a small YAC, has been assembled in the region around 80 cM of Arabidopsis thaliana chromosome 2. Clones from this contig will facilitate gene isolation in the region and can be used directly as substrates for DNA sequencing.
Conserved expression of a TASSELSEED2 homolog in the tapetum of the dioecious Silene latifolia and Arabidopsis thaliana
Authors: Lebel‐Hardenack Sabine , Ye De , Koutnikova Hana , Saedler Heinz , Grant Sarah
Published: October 2010

To investigate the genetics of male sex determination and stamen development in the dioecious plant Silene latifolia (white campion), male‐specific transcripts were isolated from developing flowers by cDNA subtraction. One of the cDNAs identified, STA1, had high DNA and amino acid sequence homology to the male sex determining gene of Zea mays (maize), TASSELSEED2. Both genes are expressed in male and not in female flowers, However, they do not share the same expression pattern. The TASSELSEED2 gene product is expressed in the gynoecium primordia of male maize flowers where it is necessary for pistil abortion. STA1 is not expressed in the gynoecium primordia of male white campion and therefore its gene product cannot perform the same function in sex determination that TASSELSEED2 performs in maize. STA1 is expressed in tapetal cells of white campion male flowers and of white campion hermaphroditic mutants. A homologous gene is also expressed in the tapetum of hermaphroditic Silene species. Tapetal expression of a homologous gene (named ATA1) was also found in Arabidopsis thaliana. The similarity in primary sequence and expression pattern of STA1 and ATA1 indicate that these genes have a conserved role in tapetum development.
Ca2+‐independent phosphorylation of a 68 kDa pollen protein is stimulated by the self‐incompatibility response in Papaver rhoeas
Authors: Rudd Jason , Franklin Christopher , Franklin‐Tong Vernonica
Published: October 2010

The self‐incompatibility (SI) response in Papaver rhoeas involves a Ca2+‐based signalling pathway, which mediates the SI‐specific inhibition of incompatible pollen. We have previously reported the identification of p26.1, a pollen protein whose phosphorylation was increased specifically as a consequence of the SI response. We have investigated whether further specific protein phosphorylation events are induced in P. rhoeas pollen. Here we report the identification of an additional pollen protein, p68, which also responds to S proteins by an increase in its phosphorylation state. This phosphorylation event occurs in living pollen tubes grown in vitro, and can be observed specifically when pollen is challenged with biologically active S proteins that are incompatible with the S alleles carried by the pollen and not when pollen was challenged with compatible S proteins. The timing of the increase in phosphorylation of p68 is temporally later than that of p26.1, occurring between 240 sec and 400 sec after challenge. This suggests that its phosphorylation is downstream of p26.1 in the SI signalling pathway(s). Surprisingly, the kinases responsible for the phosphorylation of p68 are not Ca2+‐dependent. This, and the later timing of the p68 response, suggests that a ‘second wave’ of Ca2+‐independent signalling may follow the initial Ca2+‐dependent SI signalling. This indicates that the SI signalling pathway(s) in pollen may be quite complex.
Light‐regulated expression of the pea plastocyanin gene is mediated by elements within the transcribed region of the gene
Authors: Helliwell Chris , Webster Carl , Gray John
Published: October 2010

Expression of the pea plastocyanin gene (PetE) is regulated by light in both pea and transgenic tobacco plants. However, the PetE promoter with the 5′ untranslated leader region does not direct light‐regulated expression of the GUS reporter gene in transgenic tobacco. This suggested that sequences downstream of the translation start of the PetE gene are required for light‐regulated expression. To investigate this possibility the expression of a series of chimeric gene constructs in transgenic tobacco plants was examined to assess the contributions of the promoter, the 5′ untranslated leader region, the coding region and the 3′ region of the PetE gene to light‐regulated expression. Both the coding region and the 5′ untranslated leader region of the PetE gene were found to be required for full light regulation. Full light regulation of chimeric gene constructs containing the cauliflower mosaic virus (CaMV) 35S promoter required the deletion of CaMV 5′ leader and polylinker sequences from the constructs. The presence of CaMV and polylinker sequences at the 5′ end of the PetE leader masked the light regulation directed by the transcribed region of the pea PetE gene.
The Arabidopsis MALE STERILITY 2 protein shares similarity with reductases in elongation/condensation complexes
Authors: Aarts Mark , Hodge Rachel , Kalantidis Kriton , Florack Dion , Wilson Zoe , Mulligan Bernard , Stiekema Willem , Scott Rod , Pereira Andy
Published: October 2010

The Arabidopsis thaliana MALE STERILITY 2 (MS2) gene product is involved in male gametogenesis. The first abnormalities in pollen development of ms2 mutants are seen at the stage in microsporogenesis when microspores are released from tetrads. Expression of the MS2 gene is observed in tapetum of wild‐type flowers at, and shortly after, the release of microspores from tetrads. The MS2 promoter controls GUS expression at a comparable stage in the tapetum of transgenic tobacco containing an MS2 promoter–GUS fusion. The occasional pollen grains produced by mutant ms2 plants have very thin pollen walls. They are also sensitive to acetolysis treatment, which is a test for the presence of an exine layer. The MS2 gene product shows sequence similarity to a jojoba protein that converts wax fatty acids to fatty alcohols. A possible function of the MS2 protein as a fatty acyl reductase in the formation of pollen wall substances is discussed.
Systemic elevation of phosphatidic acid and lysophospholipid levels in wounded plants
Authors: Lee Sumin , Suh Sujeoung , Kim Seju , Crain Richard , Kwak June Myoung , Nam Hong‐Gil , Lee Youngsook
Published: October 2010

Plants develop systemic defense responses upon exposure to pathogens or wounding by herbivores. Lipids and lipid metabolites have previously been implicated in induction of defense molecules during plant responses to physical wounding. Possible involvement of changes in lipid composition in systemic wound signal transduction was examined in leaves of seedlings of several different plant species. In the wounded tomato leaf, phosphatidic acid increased approximately fourfold within 5 min whereas lysophosphatidylcholine and lysophosphatidylethanolamine increased over twofold within 15 min of wounding. Similar changes in these lipids were observed in the neighboring non‐wounded leaf. In broad bean, soybean, sunflower and pepper seedlings phosphatidic acid levels increased rapidly and systemically upon wounding. The results suggest that the role of phospholipid hydrolysis and accumulation of lipid metabolites in the early events are responsible for systemic wound signal transduction in plants. Furthermore, they indicate that the wound signal propagates outside the wounded leaf within 5 min in these plants.
Genetic engineering of the multicellular green algaVolvox: a modified and multiplied bacterial antibiotic resistance gene as a dominant selectable marker
Authors: Hallmann Armin , Rappel Annette
Published: October 2010

The green alga

Volvox

represents the simplest multicellular organism:

Volvox

is composed of only two cell types, somatic and reproductive.

Volvox

, therefore, is an attractive model system for studying various aspects of multicellularity. With the biolistic nuclear transformation of

Volvox carteri

, the powerful molecular genetic manipulation of this organism has been established, but applications have been restricted to an auxotrophic mutant serving as the DNA recipient. Therefore, a dominant selectable marker working in all strains and mutants of this organism is required. Among several gene constructs tested, the most advantageous results were obtained with a chimeric gene composed of the coding sequence of the bacterial

ble

gene, conferring resistance to the antibiotic zeocin, modified with insertions of two endogenous introns from the

Volvox

arylsulfatase gene and fused to 5′ and 3′ untranslated regions from the

Volvox

β2‐tubulin gene. In the most suitable plasmid used, the gene dosage was increased 16‐fold by a technique that allows exponential multiplication of a DNA fragment. Co‐transformation of this plasmid and a non‐selectable plasmid allowed the identification of zeocin resistant transformants with nuclear integration of both selectable and non‐selectable plasmids. Stable expression of the

ble

gene and of genes from several non‐selectable plasmids is demonstrated. The modified

ble

gene provides the first dominant marker for transformation of both wild‐type and mutant strains of

Volvox

.