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Providing Mental Health and Psychosocial Support interventions (MHPSS) for forcibly displaced Ukrainians in Central and Eastern Europe poses numerous challenges due to various socio-cultural and infrastructural factors. This qualitative study explored implementation barriers reported by service providers of in-person and digital MHPSS for Ukrainian refugees displaced to Poland, Romania and Slovakia due to the war. In addition, the study aimed to generate recommendations to overcome these barriers. Semi-structured Free List and Key Informant interviews were conducted using the Design, Implementation, Monitoring and Evaluation protocol with 18 and 13 service providers, respectively. For in-person interventions, barriers included stigma, language, shortage of MHPSS providers, lack of financial aid and general lack of trust among refugees. For digital MHPSS, barriers included generational obstacles, lack of therapeutic relationships, trust issues, and lack of awareness. Recommendations included advancing public health strategies, organizational interventions, building technical literacy and support, enhancing the credibility of digital interventions and incorporating MHPSS into usual practice. By implementing the recommendations proposed in this study, policymakers, organizations and service providers can work towards enhancing the delivery of MHPSS and addressing the mental health needs of Ukrainian refugees in host countries, such as Poland, Romania and Slovakia.

Purpose: To compare T1 and T2 measurements across commercial and prototype 0.55T MRI systems in both phantom and healthy participants using the same vendor-neutral pulse sequences, reconstruction, and analysis methods., Methods: Standard spin echo measurements and abbreviated protocol measurements of T1, B1, and T2 were made on two prototype 0.55 T systems and two commercial 0.55T systems using an ISMRM/NIST system phantom. Additionally, five healthy participants were imaged at each system using the abbreviated protocol for T1, B1, and T2 measurement. The phantom measurements were compared to NMR-based reference measurements to determine accuracy, and both phantom and in vivo measurements were compared to assess reproducibility and differences between the prototype and commercial systems., Results: Vendor-neutral sequences were implemented across all four systems, and the code for pulse sequences and reconstruction is freely available. For participants, there was no difference in the mean T1 and T2 relaxation times between the prototype and commercial systems. In the phantom, there were no significant differences between the prototype and commercial systems for T1 and T2 measurements using the abbreviated protocol., Conclusion: Quantitative T1 and T2 measurements at 0.55T in phantom and healthy participants are not statistically different across the prototype and commercial systems., (© 2024 International Society for Magnetic Resonance in Medicine. This article has been contributed to by U.S. Government employees and their work is in the public domain in the USA.)

Background: A definition of the immunological features of COVID-19 pneumonia is needed to support clinical management of aged patients. In this study, we characterized the humoral and cellular immune responses in presence or absence of SARS-CoV-2 vaccination, in aged patients admitted to the IRCCS San Raffaele Hospital (Italy) for COVID-19 pneumonia between November 2021 and March 2022., Methods: The study was approved by local authorities. Disease severity was evaluated according to WHO guidelines. We tested: (A) anti-SARS-CoV-2 humoral response (anti-RBD-S IgG, anti-S IgM, anti-N IgG, neutralizing activity against Delta, BA1, BA4/5 variants); (B) Lymphocyte B, CD4 and CD8 T-cell phenotype; (C) plasma cytokines. The impact of vaccine administration and different variants on the immunological responses was evaluated using standard linear regression models and Tobit models for censored outcomes adjusted for age, vaccine doses and gender., Result: We studied 47 aged patients (median age 78.41), 22 (47%) female, 33 (70%) older than 70 years (elderly). At hospital admission, 36% were unvaccinated (VAC no ), whilst 63% had received 2 (VAC 2 ) or 3 doses (VAC 3 ) of vaccine. During hospitalization, WHO score > 5 was higher in unvaccinated (14% in VAC 3 vs. 43% in VAC 2 and 44% VACno). Independently from vaccination doses and gender, elderly had overall reduced anti-SARS-CoV-2 humoral response (IgG-RBD-S, p = 0.0075). By linear regression, the anti-RBD-S (p = 0.0060), B (p = 0.0079), CD8 (p = 0.0043) and Th2 cell counts (p = 0.0131) were higher in VAC 2 + 3 compared to VAC no . Delta variant was the most representative in VAC 2 (n = 13/18, 72%), detected in 41% of VAC no , whereas undetected in VAC 3, and anti-RBD-S production was higher in VAC 2 vs. VAC no (p = 0.0001), alongside neutralization against Delta (p = 0141), BA1 (p = 0.0255), BA4/5 (p = 0.0162). Infections with Delta also drove an increase of pro-inflammatory cytokines (IFN-α, p = 0.0463; IL-6, p = 0.0010)., Conclusions: Administration of 3 vaccination doses reduces the severe symptomatology in aged and elderly. Vaccination showed a strong association with anti-SARS-CoV-2 humoral response and an expansion of Th2 T-cells populations, independently of age. Delta variants and number of vaccine doses affected the magnitude of the humoral response against the original SARS-CoV-2 and emerging variants. A systematic surveillance of the emerging variants is paramount to define future vaccination strategies., (© 2024. The Author(s).)

Adult medulloblastoma (MB) is a rare disease affecting 0.6 persons per million adults over 19 years of age. The SHH-activated/TP53-wild type is the most common subtype, accounting for 60% of adult MBs, being characterized by mutations in PTCH1, SMO, or the TERT promoter. Several small studies demonstrate objective but short-lived responses to SMO inhibitors such as vismodegib or sonidegib. Like other oncogene-addicted solid tumors, detection of the corresponding drivers through liquid biopsy could aid in the molecular diagnosis and monitoring of the disease through less invasive procedures. However, most studies have only evaluated cerebrospinal fluid as the ctDNA reservoir, and very limited evidence exists on the role of liquid biopsy in plasma in patients with primary central nervous system tumors, including MB. We present the case of a 26-year-old patient with a recurrent MB, in which next-generation sequencing (FoundationOne CDx) revealed a mutation in PTCH1, allowing the patient to be treated with vismodegib in second line, resulting in a durable benefit lasting for 1 year. Using an in-house digital PCR probe, the PTCH1 mutation could be tracked in ctDNA during treatment with first-line chemotherapy and while on treatment with vismodegib, demonstrating a precise correlation with the radiological and clinical behavior of the disease., (© The Author(s) 2024. Published by Oxford University Press.)

Objective: To measure healthy brain [Formula: see text] and [Formula: see text] relaxation times at 0.064 T., Materials and Methods: [Formula: see text] and [Formula: see text] relaxation times were measured in vivo for 10 healthy volunteers using a 0.064 T magnetic resonance imaging (MRI) system and for 10 test samples on both the MRI and a separate 0.064 T nuclear magnetic resonance (NMR) system. In vivo [Formula: see text] and [Formula: see text] values are reported for white matter (WM), gray matter (GM), and cerebrospinal fluid (CSF) for automatic segmentation regions and manual regions of interest (ROIs)., Results: [Formula: see text] sample measurements on the MRI system were within 10% of the NMR measurement for 9 samples, and one sample was within 11%. Eight [Formula: see text] sample MRI measurements were within 25% of the NMR measurement, and the two longest [Formula: see text] samples had more than 25% variation. Automatic segmentations generally resulted in larger [Formula: see text] and [Formula: see text] estimates than manual ROIs., Discussion: [Formula: see text] and [Formula: see text] times for brain tissue were measured at 0.064 T. Test samples demonstrated accuracy in WM and GM ranges of values but underestimated long [Formula: see text] in the CSF range. This work contributes to measuring quantitative MRI properties of the human body at a range of field strengths., (© 2023. This is a U.S. Government work and not under copyright protection in the US; foreign copyright protection may apply.)

Objective: Temperature controlled T 1 and T 2 relaxation times are measured on NiCl 2 and MnCl 2 solutions from the ISMRM/NIST system phantom at low magnetic field strengths of 6.5 mT, 64 mT and 550 mT., Materials and Methods: The T 1 and T 2 were measured of five samples with increasing concentrations of NiCl 2 and five samples with increasing concentrations of MnCl 2 . All samples were scanned at 6.5 mT, 64 mT and 550 mT, at sample temperatures ranging from 10 °C to 37 °C., Results: The NiCl 2 solutions showed little change in T 1 and T 2 with magnetic field strength, and both relaxation times decreased with increasing temperature. The MnCl 2 solutions showed an increase in T 1 and a decrease in T 2 with increasing magnetic field strength, and both T 1 and T 2 increased with increasing temperature., Discussion: The low field relaxation rates of the NiCl 2 and MnCl 2 arrays in the ISMRM/NIST system phantom are investigated and compared to results from clinical field strengths of 1.5 T and 3.0 T. The measurements can be used as a benchmark for MRI system functionality and stability, especially when MRI systems are taken out of the radiology suite or laboratory and into less traditional environments., (© 2023. This is a U.S. Government work and not under copyright protection in the US; foreign copyright protection may apply.)

The coronavirus disease 2019 (COVID-19) pandemic led to the need for tracking of physical contacts and potential exposure to disease. Traditional contact tracing can be augmented by electronic tools called "electronic contact tracing" or "exposure notification.". Some methods were built to work with smartphones; however, smartphones are not prevalent in some high-contact areas (e.g., schools and nursing homes). We present the design and initial testing of low-cost, highly privacy preserving wearable exposure notification devices. Several devices were constructed based on existing hardware and operated independently of a smartphone. The method (devices and analyses) was not able to reliably use the received signal strength indicator (RSSI) as a proxy for distance between pairs of devices; the accuracy of RSSI as a proxy for distance decreased dramatically outside of the idealized conditions. However, even an imperfect device could be useful for research on how people use and move through spaces. With some improvement, these devices could be used to understand disease spread and human or animal interaction in indoor environments.

Purpose: A standard MRI system phantom has been designed and fabricated to assess scanner performance, stability, comparability and assess the accuracy of quantitative relaxation time imaging. The phantom is unique in having traceability to the International System of Units, a high level of precision, and monitoring by a national metrology institute. Here, we describe the phantom design, construction, imaging protocols, and measurement of geometric distortion, resolution, slice profile, signal-to-noise ratio (SNR), proton-spin relaxation times, image uniformity and proton density., Methods: The system phantom, designed by the International Society of Magnetic Resonance in Medicine ad hoc committee on Standards for Quantitative MR, is a 200 mm spherical structure that contains a 57-element fiducial array; two relaxation time arrays; a proton density/SNR array; resolution and slice-profile insets. Standard imaging protocols are presented, which provide rapid assessment of geometric distortion, image uniformity, T 1 and T 2 mapping, image resolution, slice profile, and SNR., Results: Fiducial array analysis gives assessment of intrinsic geometric distortions, which can vary considerably between scanners and correction techniques. This analysis also measures scanner/coil image uniformity, spatial calibration accuracy, and local volume distortion. An advanced resolution analysis gives both scanner and protocol contributions. SNR analysis gives both temporal and spatial contributions., Conclusions: A standard system phantom is useful for characterization of scanner performance, monitoring a scanner over time, and to compare different scanners. This type of calibration structure is useful for quality assurance, benchmarking quantitative MRI protocols, and to transition MRI from a qualitative imaging technique to a precise metrology with documented accuracy and uncertainty., (© 2021 The Authors. Magnetic Resonance in Medicine published by Wiley Periodicals LLC on behalf of International Society for Magnetic Resonance in Medicine.)

Many patients with recurrent/metastatic squamous cell cancer of the head and neck (SCCHN) are old or fragile and, despite deserving rapid and deep responses due to symptoms or a high tumor burden, they are not candidates for the current standard in the first-line setting of pembrolizumab plus platinum-5-FU. Other chemoimmunotherapy combinations substituting the 5-FU infusion by a taxane, may allow for less toxic effects without the need for a central venous catheter placement while maintaining efficacy. We present the case of an oral cavity cancer progressing with bulky disease to first-line cetuximab-paclitaxel in a frail and malnourished patient, where second-line treatment with pembrolizumab and reduced-dose 3-weekly carboplatin-paclitaxel achieved a deep and durable response. This is, to our knowledge, the first reported case of such combination being used in the R/M setting of SCCHN. Clinical trials should try to investigate the feasibility of this potentially less toxic and convenient combination in patients with SCCHN., (Copyright © 2021 Wolters Kluwer Health, Inc. All rights reserved.)

Magnetic resonance fingerprinting (MRF) is a powerful quantitative MRI technique capable of acquiring multiple property maps simultaneously in a short timeframe. The MRF framework has been adapted to a wide variety of clinical applications, but faces challenges in technical development, and to date has only demonstrated repeatability and reproducibility in small studies. In this review, we discuss the current implementations of MRF and their use in a clinical setting. Based on this analysis, we highlight areas of need that must be addressed before MRF can be fully adopted into the clinic and make recommendations to the MRF community on standardization and validation strategies of MRF techniques. Level of Evidence: 2 Technical Efficacy: Stage 2 J. Magn. Reson. Imaging 2020;51:675-692., (© 2019 International Society for Magnetic Resonance in Medicine.)

Low frequency nuclear magnetic resonance (NMR) is used to noninvasively and nondestructively detect spoiled tomato concentrate stored in >200 L metal-lined containers. It is shown that longitudinal and transverse NMR relaxation times change as the tomato concentrate spoils. A rapid, viscosity-dependent spoilage detection method that takes advantage of the inherent inhomogeneity in single-sided NMR instruments is proposed. Here, the effective transverse magnetization decay rate is used as a parameter to determine tomato concentrate spoilage. Three different low frequency, single-sided NMR instruments are described and compared to determine the optimum sensor for spoiled tomato concentrate detection in large-format, metal-lined, aseptic containers. The most effective NMR sensor for this application is temperature stable and has large magnetic field gradients and a homogeneous magnetic field region offset >0.5 cm from the magnet surface. PRACTICAL APPLICATION: This manuscript describes a noninvasive and nondestructive tomato concentrate spoilage detector for application to large-format, sealed, commercial storage bins., (© 2019 Institute of Food Technologists®.)

Objective: To estimate the prevalence of amyloid positivity, defined by positron emission tomography (PET)/cerebrospinal fluid (CSF) biomarkers and/or neuropathological examination, in primary progressive aphasia (PPA) variants., Methods: We conducted a meta-analysis with individual participant data from 1,251 patients diagnosed with PPA (including logopenic [lvPPA, n = 443], nonfluent [nfvPPA, n = 333], semantic [svPPA, n = 401], and mixed/unclassifiable [n = 74] variants of PPA) from 36 centers, with a measure of amyloid-β pathology (CSF [n = 600], PET [n = 366], and/or autopsy [n = 378]) available. The estimated prevalence of amyloid positivity according to PPA variant, age, and apolipoprotein E (ApoE) ε4 status was determined using generalized estimating equation models., Results: Amyloid-β positivity was more prevalent in lvPPA (86%) than in nfvPPA (20%) or svPPA (16%; p < 0.001). Prevalence of amyloid-β positivity increased with age in nfvPPA (from 10% at age 50 years to 27% at age 80 years, p < 0.01) and svPPA (from 6% at age 50 years to 32% at age 80 years, p < 0.001), but not in lvPPA (p = 0.94). Across PPA variants, ApoE ε4 carriers were more often amyloid-β positive (58.0%) than noncarriers (35.0%, p < 0.001). Autopsy data revealed Alzheimer disease pathology as the most common pathologic diagnosis in lvPPA (76%), frontotemporal lobar degeneration-TDP-43 in svPPA (80%), and frontotemporal lobar degeneration-TDP-43/tau in nfvPPA (64%)., Interpretation: This study shows that the current PPA classification system helps to predict underlying pathology across different cohorts and clinical settings, and suggests that age and ApoE genotype should be considered when interpreting amyloid-β biomarkers in PPA patients. Ann Neurol 2018;84:737-748., (© 2018 American Neurological Association.)

Chemical cues from sessile hosts can attract mobile and associated organisms and they are also impotant to maintain associations and overall biodiversity, but the identity and molecular structures of these chemicals have been little explored in the marine environment. Secondary metabolites are recognized as possible chemical mediators in the association between species of Laurencia and Aplysia, but the identity of the compounds has not been established. Here, for the first time, we experimentally verify that the sesquiterpene (+)-elatol, a compound produced by the red seaweed Laurencia dendroidea, is a chemical cue attracting the associated sea hare Aplysia brasiliana. In addition to revealing the nature of the chemical mediation between these two species, we provide evidence of a chemical cue that allows young individuals of A. brasiliana to live in association with L. dendroidea. This study highlights the importance of chemical cues in Laurencia-Aplysia association.

The significant gap between quantitative and qualitative understanding of cytoskeletal function is a pressing problem; microscopy and labeling techniques have improved qualitative investigations of localized cytoskeleton behavior, whereas quantitative analyses of whole cell cytoskeleton networks remain challenging. Here we present a method that accurately quantifies cytoskeleton dynamics. Our approach digitally subdivides cytoskeleton images using interrogation windows, within which box-counting is used to infer a fractal dimension (Df ) to characterize spatial arrangement, and gray value intensity (GVI) to determine actin density. A partitioning algorithm further obtains cytoskeleton characteristics from the perinuclear, cytosolic, and periphery cellular regions. We validated our measurement approach on Cytochalasin-treated cells using transgenically modified dermal fibroblast cells expressing fluorescent actin cytoskeletons. This method differentiates between normal and chemically disrupted actin networks, and quantifies rates of cytoskeletal degradation. Furthermore, GVI distributions were found to be inversely proportional to Df , having several biophysical implications for cytoskeleton formation/degradation. We additionally demonstrated detection sensitivity of differences in Df and GVI for cells seeded on substrates with varying degrees of stiffness, and coated with different attachment proteins. This general approach can be further implemented to gain insights on dynamic growth, disruption, and structure of the cytoskeleton (and other complex biological morphology) due to biological, chemical, or physical stimuli. © 2016 Wiley Periodicals, Inc., (© 2016 Wiley Periodicals, Inc.)

Nuclear spin relaxation rates of (2) H and (139) La in LaCl3 +(2) H2 O and La(ClO4 )3 +(2) H2 O solutions were determined as a function of pressure in order to demonstrate a new NMR probe designed for solution spectroscopy at geochemical pressures. The (2) H longitudinal relaxation rates (T1 ) vary linearly to 1.6 GPa, consistent with previous work at lower pressures. The (139) La T1 values vary both with solution chemistry and pressure, but converge with pressure, suggesting that the combined effects of increased viscosity and enhanced rates of ligand exchange control relaxation. This simple NMR probe design allows experiments on aqueous solutions to pressures corresponding roughly to those at the base of the Earth's continental crust., (© 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Little is understood regarding the impact that molecular coatings have on nanoparticle dissolution kinetics and agglomerate formation in a dilute nanoparticle dispersion. Dissolution and agglomeration processes compete in removing isolated nanoparticles from the dispersion, making quantitative time-dependent measurements of the mechanisms of nanoparticle loss particularly challenging. In this article, we present in situ ultra-small-angle X-ray scattering (USAXS) results, simultaneously quantifying dissolution, agglomeration, and stability limits of silver nanoparticles (AgNPs) coated with bovine serum albumin (BSA) protein. When the BSA corona is disrupted, we find that the loss of silver from the nanoparticle core is well matched by a second-order kinetic rate reaction, arising from the oxidative dissolution of silver. Dissolution and agglomeration are quantified, and morphological transitions throughout the process are qualified. By probing the BSA-AgNP suspension around its stability limits, we provide insight into the destabilization mechanism by which individual particles rapidly dissolve as a whole rather than undergo slow dissolution from the aqueous interface inward, once the BSA layer is breached. Because USAXS rapidly measures over the entire nanometer to micrometer size range during the dissolution process, many insights are also gained into the stabilization of NPs by protein and its ability to protect the labile metal core from the solution environment by prohibiting the diffusion of reactive species. This approach can be extended to a wide variety of coating molecules and reactive metal nanoparticle systems to carefully survey their stability limits, revealing the likely mechanisms of coating breakdown and ensuing reactions.

An ultrafast synthesis method is presented for hexanethiolate-coated gold nanoclusters (d < 2 nm, <250 atoms per nanocluster), which takes only 2 min and can be easily reproduced. With two immiscible solvents, gold nanoclusters are separated from the reaction byproducts fast and easily without any need for post-synthesis cleaning.

The reported size distribution of silver nanoparticles (AgNPs) is strongly affected by the underlying measurement method, agglomeration state, and dispersion conditions. A selection of AgNP materials with vendor-reported diameters ranging from 1 nm to 100 nm, various size distributions, and biocompatible capping agents including citrate, starch and polyvinylpyrrolidone were studied. AgNPs were diluted with either deionized water, moderately hard reconstituted water, or moderately hard reconstituted water containing natural organic matter. Rigorous physico-chemical characterization by consensus methods and protocols where available enables an understanding of how the underlying measurement method impacts the reported size measurements, which in turn provides a more complete understanding of the state (size, size distribution, agglomeration, etc.) of the AgNPs with respect to the dispersion conditions. An approach to developing routine screening is also presented.

We report a fast and highly reproducible chemical synthesis method for colloidal gold nanoparticles which are negatively charged in nonpolar solvents and coated with hydrophobic organic molecules. If a hexane droplet containing charged gold nanoparticles is mixed with a larger toluene droplet, nanoparticles immediately float to the air-toluene interface and form a close-packed monolayer film. After evaporation of the solvent molecules, the monolayer film of nanoparticles can be deposited to any substrate without any limit in size. The synthesis does not require a postsynthesis cleaning step, since the two immiscible liquid phases separate the reaction byproducts from gold nanoparticles and a minimal amount of coating molecules is used.

This anthropological study critically evaluates Brazilian caregivers' symbolic production and significance of their malnourished offspring's primary teeth, as well as their own, and describes popular dental practices. From January to June 2004, ethnographic interviews of 27 poor, low-literacy mothers were conducted at a public Malnutrition Treatment Center in Fortaleza, Ceará State. Participant observation of clinical pathways and home environments supplemented the data. Content analysis was performed. Results confirm that primary teeth are imbued with cultural significance in Northeast Brazil. Mothers examine mouths, perceive signs of decay, associate primary with permanent tooth healthiness, identify ethnodental illnesses, seek assistance, and perform rituals with exfoliated teeth. The mother's motivation to care for primary teeth is sparked by her memories of past toothache and attempts to avoid stigma and discrimination. Social determinants, not mothers' beliefs or behaviors, are the most critical obstacles to effective dental care. Legitimizing lay knowledge and empowering caregivers and children can improve oral health in Northeast Brazil.

gamma-Glutamyl transpeptidases (GGTs) are essential for hydrolysis of the tripeptide glutathione (gamma-glutamate-cysteine-glycine) and glutathione S-conjugates since they are the only enzymes known to cleave the amide bond linking the gamma-carboxylate of glutamate to cysteine. In Arabidopsis thaliana, four GGT genes have been identified based on homology with animal GGTs. They are designated GGT1 (At4g39640), GGT2 (At4g39650), GGT3 (At1g69820), and GGT4 (At4g29210). By analyzing the expression of each GGT in plants containing GGT:beta-glucuronidase fusions, the temporal and spatial pattern of degradation of glutathione and its metabolites was established, revealing appreciable overlap among GGTs. GGT2 exhibited narrow temporal and spatial expression primarily in immature trichomes, developing seeds, and pollen. GGT1 and GGT3 were coexpressed in most organs/tissues. Their expression was highest at sites of rapid growth including the rosette apex, floral stem apex, and seeds and might pinpoint locations where glutathione is delivered to sink tissues to supplement high demand for cysteine. In mature tissues, they were expressed only in vascular tissue. Knockout mutants of GGT2 and GGT4 showed no phenotype. The rosettes of GGT1 knockouts showed premature senescence after flowering. Knockouts of GGT3 showed reduced number of siliques and reduced seed yield. Knockouts were used to localize and assign catalytic activity to each GGT. In the standard GGT assay with gamma-glutamyl p-nitroanilide as substrate, GGT1 accounted for 80% to 99% of the activity in all tissues except seeds where GGT2 was 50% of the activity. Protoplasting experiments indicated that both GGT1 and GGT2 are localized extracellularly but have different physical or chemical associations.

The xenobiotic monochlorobimane is conjugated to glutathione in the cytosol of Arabidopsis thaliana, transported to the vacuole, and hydrolyzed to cysteine S-bimane [Grzam, A., Tennstedt, P., Clemens, S., Hell, R. and Meyer, A.J. (2006) Vacuolar sequestration of glutathione S-conjugates outcompetes a possible degradation of the glutathione moiety by phytochelatin synthase. FEBS Lett. 580, 6384-6390]. The work here identifies gamma-glutamyl transpeptidase 4 (At4g29210, GGT4) as the first step of vacuolar degradation of glutathione conjugates. Hydrolysis of glutathione S-bimane is blocked in ggt4 null mutants of A. thaliana. Accumulation of glutathione S-bimane in mutants and in wild-type plants treated with the high affinity GGT inhibitor acivicin shows that GGT4 is required to initiate the two step hydrolysis sequence. GGT4:green fluorescent protein fusions were used to demonstrate that GGT4 is localized in the lumen of the vacuole.

Cysteine is the first organic product of sulfate assimilation and as such is the precursor of all molecules containing reduced sulfur including methionine, glutathione, and their many metabolites. In plants, 5'-adenylylsulfate (APS) reductase is hypothesized to be a key regulatory point in sulfate assimilation and reduction. APS reductase catalyzes the two-electron reduction of APS to sulfite using glutathione as an electron donor. This paper reviews the experimental basis for this hypothesis. In addition, the results of an experiment designed to test the hypothesis by bypassing the endogenous APS reductase and its regulatory mechanisms are described. Two different bacterial assimilatory reductases were expressed in transgenic Zea mays, the thioredoxin-dependent APS reductase from Pseudomonas aeruginosa and the thioredoxin-dependent 3'-phosphoadenylylsulfate reductase from Escherichia coli. Each of them was placed under transcriptional control of the ubiquitin promoter and the protein products were targeted to chloroplasts. The leaves of transgenic Z. mays lines showed significant accumulation of reduced organic thiol compounds including cysteine, gamma-glutamylcysteine, and glutathione; and reduced inorganic forms of sulfur including sulfite and thiosulfate. Both bacterial enzymes appeared to be equally capable of deregulating the assimilative sulfate reduction pathway. The reduced sulfur compounds accumulated to such high levels that the transgenic plants showed evidence of toxicity. The results provide additional evidence that APS reductase is a major control point for sulfate reduction in Z. mays.

Homoserine kinase (HSK) produces O-phospho-l-homoserine (HserP) used by cystathionine gamma-synthase (CGS) for Met synthesis and threonine synthase (TS) for Thr synthesis. The effects of overexpressing Arabidopsis thaliana HSK, CGS, and Escherichia coli TS (eTS), each controlled by the 35S promoter, were compared. The results indicate that in Arabidopsis Hser supply is the major factor limiting the synthesis of HserP, Met and Thr. HSK is not limiting and CGS or TS control the partitioning of HserP. HSK overexpression had no effect on the level of soluble HserP, Met or Thr, however, when treated with Hser these plants produced far more HserP than wild type. Met and Thr also accumulated markedly after Hser treatment but the increase was similar in HSK overexpressing and wild-type plants. CGS overexpression was previously shown to increase Met content, but had no effect on Thr. After Hser treatment Met accumulation increased in CGS-overexpressing plants compared with wild type, whereas HserP declined and Thr was unaffected. Arabidopsis responded differentially to eTS expression depending on the level of the enzyme. At the highest eTS level the Thr content was not increased, but the phenotype was negatively affected and the T1 plants died before reproducing. Comparatively low eTS did not affect phenotype or Thr/Met level, however after Hser treatment HserP and Met accumulation were reduced compared with wild type and Thr was increased slightly. At intermediate eTS activity seedling growth was retarded unless Met was supplied and CGS expression was induced, indicating that eTS limited HserP availability for Met synthesis.

The primary flavor compound in potato, methional, is synthesized from methionine by the Strecker degradation reaction. A major problem associated with potato processing is the loss of methional. Methional or its precursor, methionine, is not added back during potato processing due to high costs of production. A novel approach to enhance the methional level in processed potato would be to increase the production of its precursor, soluble methionine (Met). Cystathionine gamma-synthase (CGS) is a key enzyme regulating methionine biosynthesis in plants. To increase the level of soluble methionine in potato, Arabidopsis thaliana CGS cDNA was introduced under transcriptional control of the cauliflower mosaic virus 35S promoter into Russet Burbank potato by Agrobacterium-mediated transformation. Ten different transgenic potato lines (CGS1-10) were analyzed. Immunoblot analysis demonstrated that Arabidopsis CGS is expressed in the leaves, tubers, and roots of transgenic potato plants. CGS enzymatic activity was higher in the leaves and roots of the transgenic potato lines compared to the wild-type potato. Methionine levels in the leaves, roots and tubers of transgenic potato lines were enhanced as high as 6-fold compared to those in wild type potato plants. The methional level in baked tubers of field-grown transgenic potato lines was increased between 2.4- and 4.4-fold in lines CGS1, CGS2, and CGS4. The increase observed in methional levels correlated with the soluble methionine level in the tubers from the same lines measured before processing. These results provide the first evidence that the methional level can be enhanced in processed potatoes by increasing the production of its precursor, methionine.

The committing step in Met and S-adenosyl-L-Met (SAM) synthesis is catalyzed by cystathionine gamma-synthase (CGS). Transgenic Arabidopsis plants overexpressing CGS under control of the cauliflower mosaic virus 35S promoter show increased soluble Met and its metabolite S-methyl-Met, but only at specific stages of development. The highest level of Met and S-methyl-Met was observed in seedling tissues and in flowers, siliques, and roots of mature plants where they accumulate 8- to 20-fold above wild type, whereas the level in mature leaves and other tissues is no greater than wild type. CGS-overexpressing seedlings are resistant to ethionine, a toxic Met analog. With these properties the transgenic lines resemble mto1, an Arabidopsis, CGS-mutant inactivated in the autogenous control mechanism for Met-dependent down-regulation of CGS expression. However, wild-type CGS was overexpressed in the transgenic plants, indicating that autogenous control can be overcome by increasing the level of CGS mRNA through transcriptional control. Several of the transgenic lines show silencing of CGS resulting in deformed plants with a reduced capacity for reproductive growth. Exogenous feeding of Met to the most severely affected plants partially restores their growth. Similar morphological deformities are observed in plants cosuppressed for SAM synthetase, even though such plants accumulate 250-fold more soluble Met than wild type and they overexpress CGS. The results suggest that the abnormalities associated with CGS and SAM synthetase silencing are due in part to a reduced ability to produce SAM and that SAM may be a regulator of CGS expression.

Sulfur is essential for life. Its oxidation state is in constant flux as it circulates through the global sulfur cycle. Plants play a key role in the cycle since they are primary producers of organic sulfur compounds. They are able to couple photosynthesis to the reduction of sulfate, assimilation into cysteine, and further metabolism into methionine, glutathione, and many other compounds. The activity of the sulfur assimilation pathway responds dynamically to changes in sulfur supply and to environmental conditions that alter the need for reduced sulfur. Molecular genetic analysis has allowed many of the enzymes and regulatory mechanisms involved in the process to be defined. This review focuses on recent advances in the field of plant sulfur metabolism. It also emphasizes areas about which little is known, including transport and recycling/degradation of sulfur compounds.

gamma-Glutamyl transpeptidases (gammaGTases) are the only enzymes known to hydrolyze the unique N-terminal amide bonds of reduced glutathione (gamma-L-glutamyl-cysteinyl-glycine), oxidized glutathione, and glutathione S-conjugates. Two gammaGTases (I and II) with K(m) values for glutathione of 110 and 90 microM were purified 2,977-fold and 2,152-fold, respectively, from ripe tomato (Lycopersicon esculentum) pericarp. Both enzymes also hydrolyze dipeptides and other tripeptides with N-terminal, gamma-linked Glu and the artificial substrates gamma-L-glutamyl-p-nitroanilide and gamma-L-glutamyl(7-amido-4-methylcoumarin). They transfer the glutamyl moiety to water or acceptor amino acids, including L-Met, L-Phe, L-Trp, L-Ala, or the ethylene precursor 1-aminocyclopropane-1-carboxylic acid. gammaGTase I and II were released from a wall and membrane fraction of a tomato fruit extract with 1.0 M NaCl, suggesting that they are peripheral membrane proteins. They were further purified by acetone precipitation, Dye Matrex Green A affinity chromatography, and hydrophobic interaction chromatography. The two gammaGTases were resolved by concanavalin A (Con A) affinity chromatography, indicating that they are differentially glycosylated. The native and SDS-denatured forms of both enzymes showed molecular masses of 43 kD.

A new conjugate, 1-(gamma-L-glutamylamino)cyclopropane-1-carboxylic acid (GACC), of the ethylene precursor 1-aminocyclopropane-1-carboxylic acid (ACC) is identified. The only previously identified conjugate of ACC is 1-(malonylamino)cyclopropane-1-carboxylic acid (MACC). GACC, not MACC, was the major conjugate formed by crude protein extracts of tomato (Lycopersicon esculentum Mill cv Ailsa Craig) fruit pericarp and seeds incubated with [14C]ACC. GACC was resolved from [14C]ACC and [14C]MACC by reversed-phase C18 thin-layer chromatography and subsequently detected and quantified using a radioisotope-imaging system. Proteins precipitated from crude extracts failed to catalyze formation of GACC unless the supernatant was added back. Reduced glutathione, but not other reducing agents, replaced the crude supernatant. When [35S-cysteine]glutathione and [3H-2-glycine]glutathione were used as substrates, neither radiolabeled glycine nor cysteine from the glutathione tripeptide was incorporated into GACC. Oxidized glutathione, S-substituted glutathione, and di- and tripeptides having an N-terminal gamma-L-glutamic acid, but lacking cysteine and glycine, also served as substrates for GACC formation. Peptides lacking the N-terminal gamma-L-glutamic acid did not serve as substrates. Acid hydrolysis of GACC yielded ACC, suggesting that GACC is an amide-linked conjugate of ACC. Taken together, these results indicate that GACC is 1-(gamma-glutamylamino)cyclopropane-1-carboxylic acid and that its formation is catalyzed by a gamma-glutamyltranspeptidase. Gas chromatography-mass spectrometry analysis of the N-acetyl dimethyl ester of GACC confirmed this structure.

1-Aminocyclopropane-1-carboxylic acid (ACC) can be oxidized to ethylene or diverted to the conjugate 1-(malonylamino)cyclopropane-1-carboxylic acid (MACC) by an ACC N-malonyltransferase. We developed a facile assay for the ACC N-malonyltransferase that resolved [14C]MACC from [14C]ACC by thin-layer chromatography and detected and quantified them using a radioisotope-imaging system. Using this assay, we showed that ACC N-malonyltransferase activity has developmental and tissue-specific patterns of expression in tomato (Lycopersicon esculentum) fruit. In the pericarp, activity was elevated for several days postanthesis, subsequently declined to a basal level, increased 3-fold at the onset of ripening, and again declined in overripe fruit. In the seed, activity increased throughout embryogenesis, maturation, and desiccation. Treatment of fruit with ethylene increased activity 50- to 100-fold in the pericarp. ACC N-malonyltransferase was purified 22,000-fold to a specific activity of 22,000 nmol min-1 mg-1 protein using ammonium sulfate precipitation, DyeMatrex Green A affinity, anion-exchange, Cibacron Blue 3GA affinity, hydrophobic interaction, and molecular filtration chromatography. Native and sodium dodecyl sulfate-denatured enzyme showed molecular masses of 38 kD, indicating that the enzyme exists as a monomer. The enzyme exhibited a Km for ACC of 500 [mu]M, was not inhibited by D- or L-amino acids, and did not conjugate [alpha]-aminoisobutyric acid or L-amino acids.

The latex of the commercial rubber tree, Hevea brasiliensis, was fractionated by ultracentrifugation as described by G. F. J. Moir ([1959] Nature 184: 1626-1628) into a top layer of rubber particles, a cleared cytoplasm, and a pellet that contains primarily specialized vacuoles known as lutoids. The proteins in each fraction were resolved by two-dimensional gel electrophoresis. Both the pellet fraction and cleared cytoplasm contained large amounts of relatively few proteins, suggesting that laticifers serve a very specialized function in the plant. More than 75% of the total soluble protein in latex was found in the pellet fraction. Twenty-five percent of the protein in the pellet was identified as chitinases/lysozymes, which are capable of degrading the chitin component of fungal cell walls and the peptidoglycan component of bacterial cell walls. Both the chitinase and lysozyme activities were localized exclusively in the pellet or lutoid fraction. The chitinases/lysozymes were resolved into acidic and basic classes of proteins and further purified. An acidic protein (molecular mass 25.5 kD) represented 20% of the chitinase activity in latex; this protein lacked the low level of lysozyme activity that is associated with many plant chitinases. Six basic proteins, having both chitinase and lysozyme activities in various ratios and molecular mass of 27.5 or 26 kD, were resolved. Two of the basic proteins had very high lysozyme specific activities which were comparable to the specific activities reported for animal lysozymes. Like animal lysozymes, but unlike previously characterized plant chitinases/lysozymes, these basic chitinases/lysozymes were also capable of completely lysing or clearing suspensions of bacterial cell walls. These results suggest that laticifers may serve a defensive role in the plant.

Platelet membrane glycoprotein IIb-IIIa exists as a calcium-dependent complex of two large peptides (designated IIb and IIIa) in Triton X-100 solutions, but it remains unknown if these peptides are subunits of one glycoprotein or are actually two individual glycoproteins in the intact platelet membrane. We used crossed immunoelectrophoresis to define the epitopes of two monoclonal antibodies to IIb-IIIa, then used these antibodies to study the structural and functional organization of IIb and IIIa in the platelet membrane. Human platelets solubilized in Triton X-100 were electrophoresed through an intermediate gel containing 125I-monoclonal IgG, then into an upper gel containing rabbit anti-human platelet antibodies. Our previously characterized antibody. Tab, and a new monoclonal antibody, T10, both bound to the immunoprecipitate corresponding to the IIb-IIIa complex. When platelets were electrophoresed after solubilization in 5 mM EDTA, 125I-Tab bound to the dissociated IIb polypeptide, but not to IIIa. In contrast, 125-I-T10 did not react with either IIb or IIIa. Thus, Tab recognizes a determinant on IIb, while T10 recognizes a determinant created only after the association of IIb and IIIa. Gel-filtered platelets from six normal donors bound 50,600 +/- 5,600 125I-T10 molecules/platelet and 47,800 +/- 11,200 125I-Tab molecules/platelet, consistent with IIb-IIIa being a heterodimer. 125I-T10 binding was identical in unactivated platelets and platelets stimulated with 10 microM ADP. However, platelets did not aggregate or bind 125I-fibrinogen until ADP was added. T10, but not Tab or nonimmune mouse antibody, inhibited ADP-induced platelet aggregation and 125I-fibrinogen binding. Our findings suggest that IIb and IIIa exist as subunits of a single membrane glycoprotein in unstimulated platelets. Fibrinogen binding appears to require not only the interaction of IIb and IIIa, but also some additional change occurring after platelet activation.

Rearrangements of membrane glycoproteins are believed to occur during platelet activation, but these changes have not been well defined. We have developed a monoclonal antibody, named S12, which demonstrates dramatically enhanced binding to platelets after thrombin activation. Unstimulated gel-filtered platelets from 12 normal individuals bound only 800 +/- 470 (S.D.) 125I-S12 molecules/cell, while platelets stimulated with 0.5 unit/ml of thrombin bound 9,600 +/- 2,600 molecules/cell (KD = 1.5 nM). Increasing thrombin concentrations produced similar increases in platelet 125I-S12 binding and [14C]serotonin secretion. S12 binding was not dependent on divalent cations. ADP and epinephrine, which caused no [14C]serotonin secretion, had little or no effect on S12 binding. We isolated the S12 binding protein by affinity chromatography of Lubrol PX-solubilized human platelet membranes on S12-agarose. When analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the isolated protein stains with both periodic acid-Schiff and Coomassie Blue and has an apparent molecular weight of 138,000 (unreduced) and 148,000 (reduced). After radioiodination of intact platelets the protein was also labeled, with apparently equal intensity in both control and thrombin-stimulated cells. The protein's staining, radiolabeling properties, and mobility on sodium dodecyl sulfate gels relative to glycoprotein IIb-IIIa fit previously defined criteria for membrane glycoprotein IIa. Our studies provide further evidence for alterations in membrane glycoproteins after platelet stimulation and suggest that S12 may serve as a useful probe of in vivo platelet activation.

Ribulose 1,5-bisphosphate carboxylase from Rhodospirillum rubrum requires CO2 and Mg2+ for activation of both CO2, both the carboxylase and oxygenase activities are stimulated by 6-phoshpo-D-gluconate, fructose 1,6-bisphosphate, 2-phosphoglycolate, 3-phosphoglycerate, NADPH, and fructose 6-phosphate. The carboxylase activity is not activated by ribose 5-phosphate. The substrate, ribulose bisphosphate, neither activates nor inhibits the CO2 and Mg2+ activation of this enzyme. Activation by CO2 and Mg2+ is rapid and results in increased susceptibility to active-site-directed protein modification reagents. Because the R. rubrum carboxylase-oxygenase is a dimer of large subunits and contains no small subunits, these results suggest that the effector binding sites of the higher plant enzyme may also be found on the large subunit.