Filter-aided sample preparation with dimethyl labeling to identify and quantify milk fat globule membrane proteins.

Bovine milk is a major nutrient source in many countries and it is produced at an industrial scale. Milk is a complex mixture of proteins, fats, carbohydrates, vitamins and minerals. The composition of the bovine milk samples can vary depending on the genetic makeup of the bovine species as well as environmental factors. It is therefore important to study the qualitative and quantitative differences of bovine milk samples. Proteins in milk can be present in casein micelles, in the serum (the water soluble fraction) or in fat globules. These fat globules have a double membrane layer with proteins being bound to or being incapsulated in the membrane layer. The identification and molecular composition of the milk proteins have gained increased interest in recent years. Proteomic techniques make it now possible to identify up to many thousands of proteins in one sample, however quantification of proteins is as yet not straightforward. We analyzed the proteins of the milk fat globule membrane using dimethyl labeling methods combined with a filter-aided sample preparation protocol. Using these methods, it is now possible to quantitatively study the detailed protein composition of many milk samples in a short period of time.

Ethnicity and dangerousness criteria for court ordered admission to a psychiatric hospital.

BACKGROUND: Black and minority ethnic (BME) populations are disproportionately detained in psychiatric hospitals. AIM: To examine the dangerousness criteria for compulsory court ordered admission to a psychiatric hospital in White and BME persons. METHOD: We examined the psychiatric examinations for court ordered compulsory admissions in 506 White and 299 BME persons from October 2004 until January 2008 in Rotterdam, the Netherlands. The White and BME groups are compared using Chi-square tests and in case of significant differences with logistic regression models adjusted for age, gender, mental disorders and socio-economic background. RESULTS: In BME persons, violence towards others and neglect of relatives were more often reasons to request court order admission as compared with Whites (39.8 vs. 25.3%, P < 0.001, respectively, 6.4 vs. 2.4%, P = 0.01). This remained true after adjustment for age, gender, mental disorders and socio-economic background [OR 1.56 (95% CI 1.12-2.18), P = 0.01, respectively; OR 3.08 (95% CI 1.31-7.26), P = 0.01]. The other reasons for a request of court order admission had a similar prevalence in both groups (suicide or self-harm, social decline, severe self-neglect, arousal of aggression of others, danger to the mental health of others, and the general safety of persons and goods). CONCLUSION: Violence towards others and neglect of relatives are more often a reason to request court ordered admission in BME than in White persons. BME patients are more often perceived as potentially dangerous to others.

[Independent psychiatrists rarely dispute an application for compulsory admission]. / Bijna altijd toekennen en weinig twijfel: overwegingen van onafhankelijke psychiaters tijdens de beoordeling rechterlijke machtiging.

BACKGROUND: In recent years in the Netherlands there has been a marked increase in the number of compulsory admissions, particularly those that require court authorisation. Little is known about the decision-making process that precedes the issuing of a court authorisation for compulsory admission. AIM: To obtain more insight into the factors that an independent psychiatrist has to consider when assessing whether he or she should sign a medical certificate that will advise on compulsory admission. METHOD: Data on clinical and demographic patient characteristics were gathered for 862 commitment applications. Motives for rejection of the application or doubt about the necessity of commitment were collected. results In the case of 9% of the applications, the psychiatrist hesitated about the need for compulsory admission but nevertheless signed the necessary medical certificate. In the case of 3% of the applications, the psychiatrist turned down the application for compulsory admission. The psychiatrist found to reject or query an application less often if a patient presented a direct physical threat to himself or others. The principal reason for rejecting an application for compulsory admission was the possibility that an alternative type of treatment was available. CONCLUSION: In principle the independent psychiatrist nearly always signs a medical certificate if the clinician treating the patient had requested a court authorisation. Factors that might help to reduce the number of court authorisations are better and earlier use of intensive care services, improved management or the deployment of legal restraints to prevent danger.

[Trends in the utilization of Dutch mental health services by older adults between 1990-2004]. / Trends in het gebruik van geestelijke gezondheidszorg door ouderen in de periode 1990-2004.

AIM: Because the Dutch population has a growing number of older people, an increasing burden on mental health services is expected. To facilitate policy making for the future, it is important to know what changes there have been in use of mental health services by elderly in the past. This study investigates changes in the use of mental health services by older adults in the period 1990-2004. METHODS: Information about the use of mental health services by older adults was retrieved from the Dutch Psychiatric Case Registers. Population size in these register areas and the unit costs of the different mental health services were taken into account. RESULTS: In total there was an increase in the number of older adults that used mental health services in the period mentioned above. The costs, however, showed a decrease, which was caused by the decrease of expensive inpatient care and the increase of less expensive outpatient care. This was mainly the case until 2002. From this year on the ratio between inpatient and outpatient care stabilized. CONCLUSION: Deinstitutionalization of mental health care for older adults was shown in the period 1990-2002. This means that expensive inpatient care is partly replaced by less expensive outpatient care. As a consequence more older adults can be treated with no rise in costs. Since 2002 deinstitutionalization came to a halt. Because a growing number of older adults will be using mental health services in the future, new forms of outpatient care should be explored.

A Tactile Seat for Direction Coding in Car Driving: Field Evaluation.

This in-traffic, field study examined the merit of using a car seat instrumented with tactile stimulation elements (tactors) to communicate directional information to a driver. A car seat fitted with an 8 times 8 matrix of tactors embedded in the seat pan was used to code eight different directions (the four cardinal and four oblique directions). With this seat mounted in a car, a field study was conducted under both smooth road and brick road vibratory conditions. The primary performance measures were directional accuracy and reaction time, measured under both alerted and simulated surprise conditions. Overall, the results show that the tactile chair seat provides a promising and robust method of providing directional information. The percentage of correct directional responses was very high (92 percent of all trials), and incorrect responses were typically just one location segment (45 degrees) off.

Exploring continuity of care in patients with alcohol use disorders using time-variant measures.

BACKGROUND/AIMS: We used time-variant measures of continuity of care to study fluctuations in long-term treatment use by patients with alcohol-related disorders. METHODS: Data on service use were extracted from the Psychiatric Case Register for the Rotterdam Region, The Netherlands. Continuity measures were calculated for each day over a 2-year observation period. Repeated measures analysis was used to identify factors that influence continuity of care over time. RESULTS: Continuity of care was higher for patients with more severe disorders. Though quantity of care was high for patients with long problem history during the first year of treatment, it decreased strongly in the second year. The intervals between treatment contacts were shorter for women, especially young ones, than for men. CONCLUSION: Time-variant measures showed differences in continuity of care that would not have been revealed if more aggregated measures of service use had been used.

Trends in het gebruik van geestelijke gezondheidszorg door ouderen in de periode 1990-2004.

TRENDS IN THE UTILIZATION OF DUTCH MENTAL HEALTH SERVICES BY OLDER ADULTS BETWEEN 1990-2004: AimBecause the Dutch population has a growing number of older people, an increasing burden on mental health services is expected. To facilitate policy making for the future, it is important to know what changes there have been in use of mental health services by elderly in the past. This study investigates changes in the use of mental health services by older adults in the period 1990-2004. Methods: Information about the use of mental health services by older adults was retrieved from the Dutch Psychiatric Case Registers. Population size in these register areas and the unit costs of the different mental health services were taken into account. Results: In total there was an increase in the number of older adults that used mental health services in the period mentioned above. The costs, however, showed a decrease, which was caused by the decrease of expensive inpatient care and the increase of less expensive outpatient care. This was mainly the case until 2002. From this year on the ratio between inpatient and outpatient care stabilized. Conclusion: Deinstitutionalization of mental health care for older adults was shown in the period 1990-2002. This means that expensive inpatient care is partly replaced by less expensive outpatient care. As a consequence more older adults can be treated with no rise in costs. Since 2002 deinstitutionalization came to a halt. Because a growing number of older adults will be using mental health services in the future, new forms of outpatient care should be explored.

[Continuity of care following suicide-related compulsory admission]. / Continuïteit van zorg na inbewaringstelling met suïcidedreiging.

In connection with recent articles about guidelines for dealing with persons attempting suicide, we investigated the quality and continuity of aftercare provided for patients admitted to a mental institution because they have attempted to commit suicide. 25% of patients received no aftercare at all. For most of the others aftercare began within two weeks; 50% of these patients were still receiving care nine months later. Follow-up studies are needed so that we can find out whether the absence of aftercare can cause problems for patients admitted to an institution because they have threatened to commit suicide.

Arabidopsis thaliana Somatic Embryogenesis Receptor Kinase 1 protein is present in sporophytic and gametophytic cells and undergoes endocytosis.

Arabidopsis thaliana plants expressing AtSERK1 fused to yellow-fluorescent protein were generated. Fluorescence was detected predominantly at the cell periphery, most likely the plasma membrane, of cells in ovules, embryo sacs, anthers, and embryos and in seedlings. The AtSERK1 protein was detected in diverse cell types including the epidermis and the vascular bundles. In some cells, fluorescent receptors were seen in small vesicle-like compartments. After application of the fungal toxin Brefeldin A, the fluorescent receptors were rapidly internalized in the root meristem and root vascular tissue. We conclude that the AtSERK1 receptor functions in a common signalling pathway employed in both sporophytic and gametophytic cells.

The Arabidopsis SOMATIC EMBRYOGENESIS RECEPTOR KINASE 1 gene is expressed in developing ovules and embryos and enhances embryogenic competence in culture.

We report here the isolation of the Arabidopsis SOMATIC EMBRYOGENESIS RECEPTOR-LIKE KINASE 1 (AtSERK1) gene and we demonstrate its role during establishment of somatic embryogenesis in culture. The AtSERK1 gene is highly expressed during embryogenic cell formation in culture and during early embryogenesis. The AtSERK1 gene is first expressed in planta during megasporogenesis in the nucellus [corrected] of developing ovules, in the functional megaspore, and in all cells of the embryo sac up to fertilization. After fertilization, AtSERK1 expression is seen in all cells of the developing embryo until the heart stage. After this stage, AtSERK1 expression is no longer detectable in the embryo or in any part of the developing seed. Low expression is detected in adult vascular tissue. Ectopic expression of the full-length AtSERK1 cDNA under the control of the cauliflower mosaic virus 35S promoter did not result in any altered plant phenotype. However, seedlings that overexpressed the AtSERK1 mRNA exhibited a 3- to 4-fold increase in efficiency for initiation of somatic embryogenesis. Thus, an increased AtSERK1 level is sufficient to confer embryogenic competence in culture.

Expression pattern of the Arabidopsis thaliana AtEP3/AtchitIV endochitinase gene.

The carrot (Daucus carota L.) EP3 chitinase was shown to be essential for somatic embryo formation in a carrot mutant cell line. We identified the Arabidopsis thaliana (L.) Heynh. ortholog of the carrot EP3-3 chitinase gene, designated as AtEP3/AtchitIV and analyzed its expression in Arabidopsis by means of reverse transcription-polymerase chain reaction and promoter::beta-glucuronidase and luciferase fusions. As in carrot, the gene is expressed during somatic embryogenesis in "nursing" cells surrounding the embryos but not in embryos themselves. In plants, gene expression is found in mature pollen and growing pollen tubes until they enter the receptive synergid, but not in endosperm and integuments as in carrot. Post-embryonically, expression is found in hydathodes, stipules, root epidermis and emerging root hairs, indicating that the Arabidopsis chitinase may have a function that is not restricted to embryogenesis.

Role of threonines in the Arabidopsis thaliana somatic embryogenesis receptor kinase 1 activation loop in phosphorylation.

The Arabidopsis thaliana somatic embryogenesis receptor kinase 1 (AtSERK1) gene encodes a receptor-like protein kinase that is transiently expressed during embryogenesis. To determine the intrinsic biochemical properties of the AtSERK1 protein, we have expressed the intracellular catalytic domain as a glutathione S-transferase fusion protein in Escherichia coli. The AtSERK1-glutathione S-transferase fusion protein mainly autophosphorylates on threonine residues (K(m) for ATP, 4 x 10(-6) m), and the reaction is Mg(2+) dependent and inhibited by Mn(2+). A K330E substitution in the kinase domain of AtSERK1 abolishes all kinase activity. The active AtSERK1(kin) can phosphorylate inactive AtSERK1(K330E) protein, suggesting an intermolecular mechanism of autophosphorylation. The AtSERK1 kinase protein was modeled using the insulin receptor kinase as a template. On the basis of this model, threonine residues in the AtSERK1 activation loop of catalytic subdomain VIII are potential targets for phosphorylation. AtSERK1 phosphorylation on myelin basic protein and casein showed tyrosine, serine, and threonine as targets, demonstrating that AtSERK1 is a dual specificity kinase. Replacing Thr-468 with either alanine or glutamic acid not only obliterated the ability of the AtSERK1 protein to be phosphorylated but also inhibited phosphorylation on myelin basic protein and casein, suggesting that Thr-468 is essential for AtSERK-mediated signaling.

Subcellular localization and oligomerization of the Arabidopsis thaliana somatic embryogenesis receptor kinase 1 protein.

The Arabidopsis thaliana somatic embryogenesis receptor kinase 1 (AtSERK1) gene is expressed in developing ovules and early embryos. AtSERK1 is also transiently expressed during somatic embryogenesis. The predicted AtSERK1 protein contains an extracellular domain with a leucine zipper motif followed by five leucine-rich repeats, a proline-rich region, a single transmembrane region and an intracellular kinase domain. The AtSERK1 cDNA was fused to two different variants of green fluorescent protein (GFP), a yellow-emitting GFP (YFP) and a cyan-emitting GFP (CFP), and transiently expressed in both plant protoplasts and insect cells. Using confocal laser scanning microscopy it was determined that the AtSERK1-YFP fusion protein is targeted to plasma membranes in both plant and animal cells. The extracellular leucine-rich repeats, and in particular the N-linked oligosaccharides that are present on them appear to be essential for correct localization of the AtSERK1-YFP protein. The potential for dimerization of the AtSERK1 protein was investigated by measuring the YFP/CFP fluorescence emission ratio using fluorescence spectral imaging microscopy. This ratio will increase due to fluorescence resonance energy transfer if the AtSERK1-CFP and AtSERK1-YFP fusion proteins interact. In 15 % of the cells the YFP/CFP emission ratio for plasma membrane localized AtSERK1 proteins was enhanced. Yeast-protein interaction experiments confirmed the possibility for AtSERK1 homodimerization. Elimination of the extracellular leucine zipper domain reduced the YFP/CFP emission ratio to control levels indicating that without the leucine zipper domain AtSERK1 is monomeric.

Expression of the Daucus carota somatic embryogenesis receptor kinase (DcSERK) protein in insect cells.

The Daucus carota somatic embryogenesis receptor kinase (DcSERK) gene serves as marker to monitor the transition from somatic into embryogenic plant cells. To determine the intrinsic biochemical properties of the DcSERK protein, a predicted transmembrane receptor, the kinase domain was expressed as a 40-kDa his-tag fusion protein in the baculovirus insect cell system. The kinase domain fusion protein was able to autophosphorylate in vitro. Phosphoamino acid analysis of the autophosphorylated DcSERK protein revealed that it was autophosphorylated on serine and threonine residues. This is the first evidence of the biochemical characterization of a transmembrane receptor kinase from embryogenic plant cell cultures.

Lipid transfer protein: a pan-allergen in plant-derived foods that is highly resistant to pepsin digestion.

BACKGROUND: Lipid transfer proteins (LTPs) are stable and highly conserved proteins of around 10 kD. They have recently been identified as allergens in fruits of the Rosaceae family. OBJECTIVE: The aim of this study was to investigate whether the highly conserved structure of LTPs justifies a designation as a true pan-allergen, and to study the role of protein stability in allergenicity. METHODS: Thirty-eight patients with a positive skin prick test to Rosaceae fruit extracts were characterized by interviews and skin prick tests. To investigate IgE cross-reactivity between Rosaceae and non-Rosaceae LTPs, RAST and RAST inhibition as well as ELISA and ELISA inhibition were performed, using whole food extracts and purified natural and recombinant LTPs. To address the role of protein stability in the allergenicity of LTP, fruit extracts and LTPs were digested with pepsin. RESULTS: IgE antibodies to Rosaceae LTPs cross-reacted with a broad range of non-Rosaceae vegetable foods. Inhibition studies with purified natural and recombinant LTPs confirmed the role of LTP in this cross-reactivity. Many of the patients with this type of cross-reactive IgE antibodies had a clinical food allergy. In contrast to the typical birch Rosaceae cross-reactive patients, the oral allergy syndrome was frequently accompanied by more severe and systemic reactions. IgE reactivity to LTP was shown to be resistant to pepsin treatment of the allergen. CONCLUSION: LTP is a true pan-allergen with a degree of cross-reactivity comparable to profilin. Due to its extreme resistance to pepsin digestion, LTP is a potentially severe food allergen.

N-acetylglucosamine and glucosamine-containing arabinogalactan proteins control somatic embryogenesis.

In plants, complete embryos can develop not only from the zygote, but also from somatic cells in tissue culture. How somatic cells undergo the change in fate to become embryogenic is largely unknown. Proteins, secreted into the culture medium such as endochitinases and arabinogalactan proteins (AGPs) are required for somatic embryogenesis. Here we show that carrot (Daucus carota) AGPs can contain glucosamine and N-acetyl-D-glucosaminyl and are sensitive to endochitinase cleavage. To determine the relevance of this observation for embryogenesis, an assay was developed based on the enzymatic removal of the cell wall from cultured cells. The resulting protoplasts had a reduced capacity for somatic embryogenesis, which could be partially restored by adding endochitinases to the protoplasts. AGPs from culture medium or from immature seeds could fully restore or even increase embryogenesis. AGPs pretreated with chitinases were more active than untreated molecules and required an intact carbohydrate constituent for activity. AGPs were only capable of promoting embryogenesis from protoplasts in a short period preceding cell wall reformation. Apart from the increase in embryogenesis, AGPs can reinitiate cell division in a subpopulation of otherwise non-dividing protoplasts. These results show that chitinase-modified AGPs are extracellular matrix molecules able to control or maintain plant cell fate.

Lipid transfer protein: a pan-allergen in plant-derived foods that is highly resistant to pepsin digestion.

BACKGROUND: Lipid transfer proteins (LTPs) are small molecules of approximately 10 kD that demonstrate high stability. They have recently been identified as allergens in the Rosaceae subfamilies of the Prunoideae (peach, apricot, plum) and of the Pomoideae (apple). They belong to a family of structurally highly conserved proteins that are also present in non-Rosaceae vegetable foods. OBJECTIVE: The aim of this study was to investigate the cross-reactivity to non-Rosaceae LTPs, and to study the role of protein stability in allergenicity. METHODS: Thirty-eight patients with a positive SPT to Rosaceae fruit extracts enriched for LTP were characterized by interview and SPT. To investigate IgE cross-reactivity between Rosaceae and non-Rosaceae LTPs, RAST and RAST inhibition as well as ELISA and ELISA inhibition were performed, using whole food extracts and purified LTPs. Both purified natural LTPs (peach, carrot and broccoli) and Pichia pastoris recombinant LTPs (carrot and wheat) were included. Pepsin digestion was used to address the role of stability in the allergenicity of LTPs. RESULTS: IgE antibodies to Rosaceae LTPs reacted to a broad range of vegetable foods, including Gramineae (cereals), Leguminosae (peanut), Juglandaceae (walnut), Anacardiaceae (pistachio), Brassicaceae (broccoli), Umbelliferae (carrot, celery), Solanaceae (tomato), Cucurbitaceae (melon), and Actinidiaceae (kiwi). Binding and inhibition studies with purified natural and recombinant LTPs confirmed their role in this cross-reactivity. Many of these cross-reactivities were accompanied by clinical food allergy, frequently including systemic reactions. Antibody binding to LTP was shown to be resistant to pepsin treatment of whole extract or purified LTP. CONCLUSION: LTP is a pan-allergen with a degree of cross-reactivity comparable to profilin. Due to its extreme resistance to pepsin digestion, LTP is a potentially severe food allergen.

Embryogenic cells in Dactylis glomerata L. (Poaceae) explants identified by cell tracking and by SERK expression.

Single mesophyll cells in leaf explants of Dactylis glomerata L. (Dactylis) that were competent to form somatic embryos directly or through callus were identified by semi-automatic cell tracking. These competent cells were a subpopulation of small, isodiametric, cytoplasm-rich cells located close to the vascular bundles. Using whole mount in situ hybridization, we showed that a similar subpopulation of cells expressed the Somatic Embryogenesis Receptor-like Kinase (SERK) gene during the induction of embryogenic cell formation. In both leaf explants and suspension cultures, a transient pattern of SERK gene expression was found during early embryo development, up to the globular stage. In later embryo stages, SERK mRNA was present in the shoot apical meristem, scutellum, coleoptile and coleorhiza.

Somatic embryogenesis in Arabidopsis thaliana is facilitated by mutations in genes repressing meristematic cell divisions.

Embryogenesis in plants can commence from cells other than the fertilized egg cell. Embryogenesis initiated from somatic cells in vitro is an attractive system for studying early embryonic stages when they are accessible to experimental manipulation. Somatic embryogenesis in Arabidopsis offers the additional advantage that many zygotic embryo mutants can be studied under in vitro conditions. Two systems are available. The first employs immature zygotic embryos as starting material, yielding continuously growing embryogenic cultures in liquid medium. This is possible in at least 11 ecotypes. A second, more efficient and reproducible system, employing the primordia timing mutant (pt allelic to hpt, cop2, and amp1), was established. A significant advantage of the pt mutant is that intact seeds, germinated in 2,4-dichlorophenoxyacetic acid (2, 4-D) containing liquid medium, give rise to stable embryonic cell cultures, circumventing tedious hand dissection of immature zygotic embryos. pt zygotic embryos are first distinguishable from wild type at early heart stage by a broader embryonic shoot apical meristem (SAM). In culture, embryogenic clusters originate from the enlarged SAMs. pt somatic embryos had all characteristic embryo pattern elements seen in zygotic embryos, but with higher and more variable numbers of cells. Embryogenic cell cultures were also established from seedling, of other mutants with enlarged SAMs, such as clavata (clv). pt clv double mutants showed additive effects on SAM size and an even higher frequency of seedlings producing embryogenic cell lines. pt clv double mutant plants had very short fasciated inflorescence stems and additive effects on the number of rosette leaves. This suggests that the PT and CLV genes act in independent pathways that control SAM size. An increased population of noncommitted SAM cells may be responsible for facilitated establishment of somatic embryogenesis in Arabidopsis.

A leucine-rich repeat containing receptor-like kinase marks somatic plant cells competent to form embryos.

The first somatic single cells of carrot hypocotyl explants having the competence to form embryos in the presence of 2,4-dichlorophenoxyacetic acid (2,4-D) were identified using semi-automatic cell tracking. These competent cells are present as a small subpopulation of enlarged and vacuolated cells derived from cytoplasm-rich and rapidly proliferating non-embryogenic cells that originate from the provascular elements of the hypocotyl. A search for marker genes to monitor the transition of somatic into competent and embryogenic cells in established suspension cell cultures resulted in the identification of a gene transiently expressed in a small subpopulation of the same enlarged single cells that are formed during the initiation of the embryogenic cultures from hypocotyl explants. The predicted amino acid sequence and in vitro kinase assays show that this gene encodes a leucine-rich repeat containing receptor-like kinase protein, designated Somatic Embryogenesis Receptor-like Kinase (SERK). Somatic embryos formed from cells expressing a SERK promoter-luciferase reporter gene. During somatic embryogenesis, SERK expression ceased after the globular stage. In plants, SERK mRNA could only be detected transiently in the zygotic embryo up to the early globular stage but not in unpollinated flowers nor in any other plant tissue. These results suggest that somatic cells competent to form embryos and early globular somatic embryos share a highly specific signal transduction chain with the zygotic embryo from shortly after fertilization to the early globular embryo.