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Background: ASTHMAXcel PRO, an enhanced version of the ASTHMAXcel mobile application, has been developed to deliver comprehensive, guideline-based asthma education while also facilitating the collection of patient-reported outcomes (PROs) and enhancing user experience., Objective: To perform field testing and conduct formative and summative evaluation of the ASTHMAXcel PRO application to assess its impact on patient satisfaction, usability, and usage., Methods: Twenty-eight adult patients completed a baseline visit during which ASTHMAXcel PRO was introduced, health literacy was assessed, and demographic data were collected. They were instructed to use the app for 4 weeks. The Questionnaire for User Interface Satisfaction (QUIS) and the Unified Theory of Acceptance and Use of Technology (UTAUT) questionnaire were administered at baseline and 4 weeks to assess user satisfaction and technology acceptance, respectively. Semi-structured interviews were conducted to gather feedback regarding the application from patients., Results: The baseline total scores were high for both UTAUT and QUIS (mean (SD): 64.2 (10.1), 6.8 (2.2) respectively) indicating that user satisfaction and acceptance began at high levels. UTAUT total score, as well as all domain scores, improved significantly from baseline to 4 weeks ( p < 0.02). QUIS total score along with several domain scores (screen, system capabilities, usability) also increased from baseline to 4-weeks ( p = 0.03, 0.01, 0.03, 0.01, respectively). These improvements remained significant when adjusting for age, gender, education, and health literacy. Patients reported that the application was helpful, informative, and easy to understand and use., Conclusion: The significant increases in satisfaction and technology adoption observed among ASTHMAXcel PRO users demonstrate that the application is viable and has the potential to improve upon usability challenges faced by existing mobile health applications.

Background: Cyclic vomiting syndrome (CVS) is an enigmatic and debilitating disorder of gut-brain interaction that is characterized by recurrent episodes of severe vomiting and nausea. It significantly impairs patients' quality of life and can lead to frequent medical visits and substantial health care costs. The diagnosis for CVS is often protracted and complex, primarily due to its exclusionary diagnosis nature and the lack of specific biomarkers. This typically leads to a considerable delay in accurate diagnosis, contributing to increased patient morbidity. Additionally, the absence of approved therapies for CVS worsens patient hardship and reflects the urgent need for innovative, patient-centric solutions to improve CVS management., Objective: We aim to develop a digital patient assistant (DPA) for patients with CVS to address their unique needs, and iteratively enhance the technical features and user experience on the initial DPA versions., Methods: The development of the DPA for CVS used a design thinking approach, prioritizing user needs. A literature review and Patient Advisory Board shaped the initial prototype, focusing on diagnostic support and symptom tracking. Iterative development, informed by the design thinking approach and feedback from patients with CVS and caregivers through interviews and smartphone testing, led to significant enhancements in user interaction and artificial intelligence integration. The final DPA's effectiveness was validated using the System Usability Scale and feedback questions, ensuring it met the specific needs of the CVS community., Results: The DPA developed for CVS integrates an introductory bot, daily and weekly check-in bots, and a knowledge hub, all accessible via a patient dashboard. This multicomponent solution effectively addresses key unmet needs in CVS management: efficient symptom and impacts tracking, access to comprehensive disease information, and a digital health platform for disease management. Significant improvements, based on user feedback, include the implementation of artificial intelligence features like intent recognition and data syncing, enhancing the bot interaction and reducing the burden on patients. The inclusion of the knowledge hub provides educational resources, contributing to better disease understanding and management. The DPA achieved a System Usability Scale score of 80 out of 100, indicating high ease of use and relevance. Patient feedback highlighted the DPA's potential in disease management and suggested further applications, such as integration into health care provider recommendations for patients with suspected or confirmed CVS. This positive response underscores the DPA's role in enhancing patient engagement and disease management through a patient-centered digital solution., Conclusions: The development of this DPA for patients with CVS, via an iterative design thinking approach, offers a patient-centric solution for disease management. The DPA development framework may also serve to guide future patient digital support and research scenarios., (©Gaurav Narang, Yaozhu J Chen, Nicole Wedel, Melody Wu, Michelle Luo, Ashish Atreja. Originally published in JMIR Formative Research (https://formative.jmir.org), 06.06.2024.)

The total number of confirmed cases of COVID-19 caused by SARS-CoV-2 virus infection is over 621 million. Post-COVID-19 syndrome, also known as long COVID or long-haul COVID, refers to a persistent condition where individuals experience symptoms and health issues after the acute phase of COVID-19. The aim of this study was to assess the strength and fatigue of skeletal muscles in people recovered from COVID-19. A total of 94 individuals took part in this cross-sectional study, with 45 participants (referred to as the Post-COVID Cohort, PCC) and 49 healthy age-matched volunteers (Healthy Control Cohort, HCC). This research article uses the direct dynamometry method to provide a detailed analysis of post-COVID survivors' strength and power characteristics. The Biodex System 4 Pro was utilized to evaluate muscle strength characteristics during the fatigue test. The fatigue work in extensors and flexors was significantly higher in the PCC. The PCC also showed significantly less power in both extensors and flexors compared to the HCC. In conclusion, this study provides compelling evidence of the impact of post-COVID-19 fatigue on muscle performance, highlighting the importance of considering these effects in the rehabilitation and care of individuals recovering from the virus. PCC achieved lower muscle strength values than HCC.

Trustworthy-looking faces are also perceived as more attractive, but are there other meaningful cues that contribute to perceived trustworthiness? Using data-driven models, we identify these cues after removing attractiveness cues. In Experiment 1, we show that both judgments of trustworthiness and attractiveness of faces manipulated by a model of perceived trustworthiness change in the same direction. To control for the effect of attractiveness, we build two new models of perceived trustworthiness: a subtraction model, which forces the perceived attractiveness and trustworthiness to be negatively correlated (Experiment 2), and an orthogonal model, which reduces their correlation (Experiment 3). In both experiments, faces manipulated to appear more trustworthy were indeed perceived to be more trustworthy, but not more attractive. Importantly, in both experiments, these faces were also perceived as more approachable and with more positive expressions, as indicated by both judgments and machine learning algorithms. The current studies show that the visual cues used for trustworthiness and attractiveness judgments can be separated, and that apparent approachability and facial emotion are driving trustworthiness judgments and possibly general valence evaluation.

Introduction: The total number of confirmed cases of COVID-19 caused by the SARS-CoV-2 virus infection is over 621 million in the world. In approximately 63% of cases, the patient still experiences persistent symptoms 30 days after the onset of symptoms or hospitalisation, and 45.9% of patients have experienced or will experience symptoms for at least three months. Despite the prevalence of chronic symptoms and pathological changes that may affect gait and functional mobility in people with a history of COVID-19, there are few publications investigating the impact of these abnormalities. This study aims to determine the long-term effects of COVID-19 on gait and the Timed-Up and Go Task., Material and Methods: A total of 30 individuals took part in the experiment. The subjects in the study group were infected with the COVID-19 virus and required hospital treatment. Prior to the study, the subjects had no chronic diseases or other conditions affecting the musculoskeletal system. The non-infected by COVID-19 group was a healthy population with no history of COVID-19 disease. The study used the inertial system wireless motion analysis system based on 15 inertial sensors (inertial measurement units, IMUs). IMU sensors were placed on the following body segments: head, sternum, middle and lower spine, shoulder, arm, forearm, hand, shank, for the left and right limb. Movement task reports generated from the recording were created using myoRESEARCH 3.10. The subjects in the study group were asked to perform a movement task test-the Timed-Up and Go Test (TUG): sit-to-stand, walk (3 m) without change in direction, walk termination, and stand-to-sit., Results: It took 46% longer for those infected by COVID-19 (participants) to complete the entire movement task compared to those in the not-infected by COVID-19 group. Sit-to-Stand Time [s] was greater in the infected by COVID-19 group and was 2.1 ± 0.7. Mean Walking Speed [m/s] was lower than in the not-infected by COVID-19 group and was 0.26 ± 0.07. Walking cadence [steps/min] was lower and was 21.2 ± 1.2. Infected by COVID-19 participants achieved a smaller anterior pelvic tilt angle ( p < 0.001) and a smaller hip flexion angle ( p = 0.025), with an increase in knee ( p < 0.001) and ankle ( p < 0.001) flexion angles., Conclusions: Individuals in the infected by COVID-19 group present changes in the ranges of motion and the time to complete the TUG task, despite the fact that at least eight weeks passed after hospital discharge.

Preeclampsia is a severe pregnancy complication with high potential for adverse effects on maternal and fetal health during the perinatal period. It is also associated with an increased risk of maternal cardiovascular disease later in life. Development of preeclampsia can be decreased by prescribing low-dose aspirin to high-risk women. At present, maternal and pregnancy factors are used to assess the risk of preeclampsia. One additional factor that could add to the assessment of risk is a family history of hypertension, cardiovascular disease, or diabetes, especially for nulliparous women who do not have a pregnancy history to inform treatment decisions. Therefore, we conducted a systematic review to assess the association between family history of the aforementioned conditions and preeclampsia. Four databases including MEDLINE, EMBASE, the Cochrane Library, and CINAHL/pre-CINAHL were searched for observational studies that examined a family history of hypertension, cardiovascular disease, or diabetes in women with preeclampsia and in a control population. Studies were evaluated for quality using the Newcastle-Ottawa Scale. A total of 84 relevant studies were identified. A meta-analysis was not conducted due to suspected heterogeneity in the included studies. Most studies reported a positive association between a family history of hypertension or cardiovascular disease and the development of preeclampsia. The majority of studies examining family history of diabetes reported non-significant associations. Overall, family history of hypertension or cardiovascular disease is associated with a higher risk for developing preeclampsia and should be considered when assessing women in the first trimester for low-dose aspirin., (Copyright © 2020. Published by Elsevier Inc.)

This article reviews the evidence and nutrition practice recommendations from the American Dietetic Association's nutrition practice guidelines for type 1 and type 2 diabetes in adults. The research literature was reviewed to answer nutrition practice questions and resulted in 29 recommendations. Here, we present the recommendations and provide a comprehensive and systematic review of the evidence associated with their development. Major nutrition therapy factors reviewed are carbohydrate (intake, sucrose, non-nutritive sweeteners, glycemic index, and fiber), protein intake, cardiovascular disease, and weight management. Contributing factors to nutrition therapy reviewed are physical activity and glucose monitoring. Based on individualized nutrition therapy client/patient goals and lifestyle changes the client/patient is willing and able to make, registered dietitians can select appropriate interventions based on key recommendations that include consistency in day-to-day carbohydrate intake, adjusting insulin doses to match carbohydrate intake, substitution of sucrose-containing foods, usual protein intake, cardioprotective nutrition interventions, weight management strategies, regular physical activity, and use of self-monitored blood glucose data. The evidence is strong that medical nutrition therapy provided by registered dietitians is an effective and essential therapy in the management of diabetes., (Copyright © 2010 American Dietetic Association. Published by Elsevier Inc. All rights reserved.)

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) of higher plants catalyzes an NADPH-consuming reaction, which is part of the Calvin cycle. This reaction is regulated by light via thioredoxins and metabolites, while a minor NADH-dependent activity is constant and constitutive. The major native isozyme is formed by A- and B-subunits in stoichiometric ratio (A2B2, A8B8), but tetramers of recombinant B-subunits (GapB) display similar regulatory features to A2B2-GAPDH. The C-terminal extension (CTE) of B-subunits is essential for thioredoxin-mediated regulation and NAD-induced aggregation to partially inactive oligomers (A8B8, B8). Deletion mutant B(minCTE) is redox insensitive and invariably tetrameric, and chimeric mutant A(plusCTE) acquired redox sensitivity and capacity to aggregate to very large oligomers in presence of NAD. Redox regulation principally affects the turnover number, without significantly changing the affinity for either 1,3-bisphosphoglycerate or NADPH. Mutant R77A of GapB, B(R77A), is down-regulated and mimics the behavior of oxidized GapB under any redox condition, whereas mutant B(E362Q) is constantly up-regulated, resembling reduced GapB. Despite their redox insensitivity, both B(R77A) and B(E362Q) mutants are notably prone to aggregate in presence of NAD. Based on structural data and current functional analysis, a model of GAPDH redox regulation is presented. Formation of a disulfide in the CTE induces a conformational change of the GAPDH with repositioning of the terminal amino acid Glu-362 in the proximity of Arg-77. The latter residue is thus distracted from binding the 2'-phosphate of NADP, with the final effect that the enzyme relaxes to a conformation leading to a slower NADPH-dependent catalytic activity.

Purpose: Lintuzumab (HuM195) is an unconjugated humanized murine monoclonal antibody directed against the cell surface myelomonocytic differentiation antigen CD33. In this study, the efficacy of lintuzumab in combination with induction chemotherapy was compared with chemotherapy alone in adults with first relapsed or primary refractory acute myeloid leukemia (AML)., Patients and Methods: Patients with relapsed or primary resistant AML (duration of first response, zero to 12 months) were randomly assigned to receive either mitoxantrone 8 mg/m(2), etoposide 80 mg/m(2), and cytarabine 1 g/m(2) daily for 6 days (MEC) in combination with lintuzumab 12 mg/m(2), or MEC alone. Overall response, defined as the rate of complete remission (CR) and CR with incomplete platelet recovery (CRp), was the primary end point of the study, with additional analyses of survival time and toxicity., Results: A total of 191 patients were randomly assigned from November 1999 to April 2001. The percent CR plus CRp with MEC plus lintuzumab was 36% v 28% in patients treated with MEC alone (P = .28). The overall median survival was 156 days and was not different in the two arms of the study. Apart from mild antibody infusion-related toxicities (fever, chills, and hypotension), no differences in chemotherapy-related adverse effects, including hepatic and cardiac dysfunction, were observed with the addition of lintuzumab to induction chemotherapy., Conclusion: The addition of lintuzumab to salvage induction chemotherapy was safe, but did not result in a statistically significant improvement in response rate or survival in patients with refractory/relapsed AML.

The chloroplast enzyme phosphoribulokinase (PRK; EC 2.7.1.19) is part of the Calvin cycle (reductive pentose phosphate pathway) responsible for CO(2) fixation in photosynthetic organisms. In green algae and vascular plants, this enzyme is light regulated via reversible reduction by reduced thioredoxin. We have sequenced and characterized the gene of the PRK from the marine diatom Odontella sinensis and found that the enzyme has the conserved cysteine residues necessary for thioredoxin-dependent regulation. Analysis of enzymatic activity of partially purified diatom enzyme and of purified protein obtained by native overexpression in Escherichia coli, however, revealed that under natural redox conditions the diatom enzyme is generally active. Treatment of the enzyme with strong oxidants results in inhibition of the enzyme, which is reversible by subsequent incubation with reducing agents. We determined the redox midpoint potentials of the regulatory cysteine in the PRK from O. sinensis in comparison to the respective spinach (Spinacia oleracea) enzyme and found a more positive redox potential for the diatom PRK, indicating that in vivo this enzyme might not be regulated by thioredoxin. We also demonstrate that in protease-treated diatom plastids, activities of enzymes of the oxidative pentose phosphate pathway are not detectable, thus reducing the need for a tight regulation of the Calvin cycle in diatoms. We discuss our results in the context of rearrangements of the subcellular compartmentation of metabolic pathways due to the peculiar evolution of diatoms by secondary endocytobiosis.

CP12 is an 8.5-kDa nuclear-encoded chloroplast protein, isolated from higher plants. It forms part of a core complex of two dimers of phosphoribulokinase (PRK), two tetramers of glyceraldehyde 3-phosphate dehydrogenase (GAPDH), and CP12. The role of CP12 in this complex assembly has not been determined. To address this question, we cloned a cDNA encoding the mature CP12 from the green alga Chlamydomonas reinhardtii and expressed it in Escherichia coli. Sequence alignments show that it is very similar to other CP12s, with four conserved cysteine residues forming two disulfide bridges in the oxidized CP12. On the basis of reconstitution assays and surface plasmon resonance binding studies, we show that oxidized, but not reduced, CP12 acts as a linker in the assembly of the complex, and we propose a model in which CP12 associates with GAPDH, causing its conformation to change. This GAPDH/CP12 complex binds PRK to form a half-complex (one unit). This unit probably dimerizes due partially to interactions between the enzymes of each unit. Reduced CP12 being unable to reconstitute the complex, we studied the structures of oxidized and reduced CP12 by NMR and circular dichroism to determine whether reduction induced structural transitions. Oxidized CP12 is mainly composed of alpha helix and coil segments, and is extremely flexible, while reduced CP12 is mainly unstructured. Remarkably, CP12 has similar physicochemical properties to those of "intrinsically unstructured proteins" that are also involved in regulating macromolecular complexes, or in their assembly. CP12s are thus one of the few protein families of intrinsically unstructured proteins specific to plants.

Light/dark modulation of the higher plant Calvin-cycle enzymes phosphoribulokinase (PRK) and NADP-dependent glyceraldehyde 3-phosphate dehydrogenase (NADP- GAPDH-A2B2) involves changes of their aggregation state in addition to redox changes of regulatory cysteines. Here we demonstrate that plants possess two different complexes containing the inactive forms (a) of NADP-GAPDH and PRK and (b) of only NADP-GAPDH, respectively, in darkened chloroplasts. While the 550-kDa PRK/GAPDH/CP12 complex is dissociated and activated upon reduction alone, activation and dissociation of the 600-kDa A8B8 complex of NADP-GAPDH requires incubation with dithiothreitol and the effector 1,3-bisphosphoglycerate. In the light, PRK is therefore completely in its activated state under all conditions, even in low light, while GAPDH activation in the light is characterized by a two-step mechanism with 60-70% activation under most conditions in the light, and the activation of the remaining 30-40% occurring only when 1,3-bisphosphoglycerate levels are strongly increasing. In vitro studies with the purified components and coprecipitation experiments from fresh stroma using polyclonal antisera confirm the existence of these two aggregates. Isolated oxidized PRK alone does not reaggregate after it has been purified in its reduced form; only in the presence of both CP12 and purified NADP-GAPDH, some of the PRK reaggregates. Recombinant GapA/GapB constructs form the A8B8 complex immediately upon expression in E. coli.

This prospective study investigated the role of reduced hepatic synthesis of regulating proteins in coagulopathy after partial hepatectomy (PH) compared with major abdominal surgery (MAS) without involvement of the liver. Furthermore, we studied the effect of rBPI21, an endotoxin-neutralizing agent, on coagulopathy after PH was studied. Compared with MAS, PH resulted in significantly elevated levels of thrombin-antithrombin-III and plasmin-alpha2-antiplasmin complexes. Levels of antithrombin-3, alpha2-antiplasmin, fibrinogen, plasminogen, alpha2-macroglobulin (alpha2-M), and C1-inhibitor remained lower following PH. Treatment with rBPI21 led to significantly lower levels of tissue-type plasminogen activator (t-PA). Post-operative disseminated intravascular coagulation (DIC) was associated with significantly higher bilirubin and t-PA plasma levels and significantly lower levels of alpha2-M. This study indicates that PH induced hepatic failure results in decreased synthesis of hepatic regulating plasma proteins and subsequent activation of coagulation and fibrinolysis. Prevention of t-PA release by rBPI21 may have important clinical implications. Decreased availability of alpha2-M may be a factor in post-operative DIC.

Background: HuM291 is a humanized anti-CD3 monoclonal antibody engineered to reduce binding to Fcgamma receptors and complement fixation. HuM291 has a long serum half-life and mediated profound depletion of circulating T cells in chimpanzees; HuM291 also has significantly less mitogenic and cytokine-releasing activity than OKT3 in vitro., Methods: A phase I dose-escalation study was conducted in 15 end-stage renal disease patients scheduled for renal allografts from living donors. Patients received one i.v. HuM291 injection before transplantation. Five doses were tested: 0.015 microg/kg, 0.15 microg/kg, 0.0015 mg/kg, 0.0045 mg/kg, and 0.015 mg/kg. Patients were followed for adverse events, laboratory abnormalities, serum cytokine levels, pharmacokinetics, and CD2+, CD3+, CD4+, and CD8+ T cell counts., Results: HuM291 was well tolerated; most adverse events were mild to moderate in severity and included headache, nausea, chills, and fever. These occurred within the first few hours after HuM291 administration, resolved within 24 to 48 hr, and were likely related to cytokine release. In general, peak tumor necrosis factor-alpha, interferon-gamma, and interleukin-6 levels were detected 1 to 6 hr postdosing only at the three highest doses and were generally undetectable by 24-hr postdosing. Serious adverse events possibly related to HuM291 included clotting of a fistula (two patients), chemical cellulitis (one patient), and increased serum creatinine/decreased hematocrit (one patient). At doses > or = 0.0015 mg/kg (0.1 mg/70 kg), HuM291 induced rapid, marked depletion of peripheral T cells within 2 hr; duration of T cell depletion was dose dependent. At the two highest dose levels, T cells remained depleted for approximately 1 week., Conclusions: A single HuM291 dose rapidly depleted circulating T cells in a dose-dependent manner and was associated with only mild to moderate symptoms of cytokine release.

Phase I pharmacokinetic and safety studies were conducted in healthy volunteers with rBPI21, a recombinant protein derived from the amino terminal domain of human bactericidal/permeability-increasing protein. rBPI21 was administered as a 30-minute infusion at doses of 0.25 to 4 mg/kg or as a 24- to 48-hour infusion at doses of 2 to 8 mg/kg. For the 30-minute infusions, the clearance of rBPI21 decreased with increasing dose from 8.4 mL/min/kg at 0.25 mg/kg to 3.3 mL/min/kg at 4 mg/kg. For rBPI21 infused over 24 to 48 hours the clearance was 10 to 11 mL/min/kg. The concentration-time profile of rBPI21 was well described by a three-compartmental model with parallel first-order and Michaelis-Menten (saturable) elimination. This model for the clearance of rBPI21 has been useful in estimating starting doses for therapeutic clinical trials.

Objective: To (a) explore the contribution of infant, environmental, and historical factors to the number of days from initiation to achievement of full nipple feeding (transition time) for premature infants with a history of lung disease; (b) examine differences in the contribution of infant and environmental factors to transition time made by historical era, either earlier (in the 1980s) or later (in the 1990s); and (c) compare, within eras, the contribution to transition time of infant and environmental factors for infants with each lung diagnosis, respiratory distress syndrome (RDS) without bronchopulmonary dysplasia (BPD) or BPD., Design: Data were collected at two midwestern hospitals from the records of premature infants with a diagnosis of either RDS without BPD or BPD. The influence on transition time of infant, environmental and historical factors was assessed with the Cox proportional hazards model. This analytic model, a form of regression analysis, also was used to explore how era influenced the contribution to transition time of infant and environmental factors. Finally, the contribution to transition time of infant and environmental factors was examined within diagnostic group for each era., Sample: The hospital records audited were for infants who were 32 weeks gestational age or less with weight appropriate for gestational age. The number in each diagnostic group for each era was (a) BPD--Early, n = 35; (b) RDS--Early, n = 21; (c) BPD--Late, n = 21; and (d) RDS--Late, n = 15)., Results: All three types of factors (infant, environmental, and historical) contributed significantly (p < .05) to shortening or lengthening transition time. A diagnosis of BPD lengthened transition time only in the early era. Across both eras, the number of days on tube feedings significantly lengthened transition time, and the older the infant in postconceptional age (PCA) at initiation of nipple feeding, the shorter the transition time., Conclusion: The contribution of infant, environmental, and historical factors to transition time confirmed the basic structure of the theoretical model of transition time for premature infants with a history of lung disease. The influence of era on the contributions to transition time of infant and environmental factors suggests that care policy and practice have shortened the transition time. Although the current findings support the basic structure of the theoretical model for infants with either RDS or BPD, the marginally significant (p < .10) shortening effect of PCA on transition time for infants with BPD in both eras suggests that advancement to full nipple feeding may be limited by neurodevelopmental capacities, including respiratory control. How these capacities can be supported for advancement to full nipple feeding is a challenge for nursing practice and research.

For higher plant chloroplasts, two key enzymes of the Calvin cycle, phosphoribulokinase (EC 2.7.1.19) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH, EC 1.2.1.13), have recently been shown to be oligomerized onto the nonenzymatic peptide CP12. Enzymatic activity depends on complex dissociation, mediated by NADPH. The discovery of genes for CP12 in mosses, green algae, and cyanobacteria, together with the analysis of equivalent multiprotein complexes of Chlamydomonas and Synechocystis suggests that light regulation of Calvin cycle activity via NADPH-mediated reversible phosphoribulokinase/CP12/GAPDH complex dissociation is conserved in all photosynthetic organisms, prokaryotes and eukaryotes. In vitro complex reconstitution assays with heterologously expressed Synechocystis wild-type and mutagenized CP12 demonstrate a conserved subunit composition, stoichiometry, and topology in this complex. Further finding of genes, coding for chimeric proteins, carrying CP12 or parts of it as genetic fusions, indicates that evolution has used the peptide loops of CP12 as universal modules to keep various enzymatic activities under the control of NADP(H). These fusion events occurred at least twice in evolution. First was the fusion of the duplicated genes for CP12 and the ORF4 protein of Anabaena variabilis to the chimeric gene for the heterocyst-specific expressed ORF3 protein, most probably involved in N2 fixation. A second gene fusion, which led to the higher plant chloroplast-specific GAPDH subunit, GAPB, has taken place during the transition from water- to land plants.

Background: Meningococcal sepsis remains an important cause of morbidity and mortality. We hypothesised that children with severe meningococcaemia might benefit from inhibition of the inflammatory processes thought responsible for fulminant disease. rBPI21 is a recombinant, N-terminal fragment of human bactericidal/permeability-increasing protein, which kills meningococci and binds to and clears bacterial endotoxin, these being the primary inducers of the systemic inflammation. The aim of this study was to determine the safety and kinetics of rBPI21 in children with severe meningococcaemia and to make a preliminary assessment of clinical outcome., Methods: In this open-label, dose-escalation, phase I/II trial in severe meningococcaemia (Glasgow meningococcal prognostic septicaemia score [GMSPS] > or = 8), 26 patients aged 1-18 years, who had received their first dose of antibiotics no more than 8 hours earlier were given rBPI21 by infusion at total doses of 1.0, 2.0, and 4.0 mg/kg., Findings: The patients had significantly raised plasma concentrations of bacterial endotoxin and cytokines. Peak and steady state BPI concentrations were comparable with pharmacokinetic data in healthy adults. All complications were compatible with the expected pattern for severe meningococcal sepsis. Only one patient died. This outcome was found to compare favourably with a predicted mortality of > or = 30% by GMSPS, > or = 15% by plasma endotoxin values, > or = 28% by plasma interleukin-6 concentrations, 29-49% by severity of coagulopathy, and 20% (11/54) by comparison with recent historical patients consecutively treated in participating centres before this study., Interpretation: This, the first clinical trial or rBPI21, shows that rBPI21 can be safely administered to children with severe meningococcaemia and that the pharmacokinetics are consistent with patterns seen in healthy adults. Predicted mortality, on the basis of GMSPS, laboratory indices of inflammation and coagulopathy, and historical controls, was for between four and eight deaths. These findings have prompted a phase III randomised trial.

CP12 is a small nuclear encoded chloroplast protein of higher plants, which was recently shown to interact with NAD(P)H-glyceraldehyde-3-phosphate dehydrogenase (GAPDH; EC 1.2.1. 13), one of the key enzymes of the reductive pentosephosphate cycle (Calvin cycle). Screening of a pea cDNA library in the yeast two-hybrid system for proteins that interact with CP12, led to the identification of a second member of the Calvin cycle, phosphoribulokinase (PRK; EC 2.7.1.19), as a further specific binding partner for CP12. The exchange of cysteines for serines in CP12 demonstrate that interaction with PRK occurs at the N-terminal peptide loop of CP12. Size exclusion chromatography and immunoprecipitation assays reveal the existence of a stable 600-kDa PRK/CP12/GAPDH complex in the stroma of higher plant chloroplasts. Its stoichiometry is proposed to be of two N-terminally dimerized CP12 molecules, each carrying one PRK dimer on its N terminus and one A2B2 complex of GAPDH subunits on the C-terminal peptide loop. Incubation of the complex with NADP or NADPH, in contrast to NAD or NADH, causes its dissociation. Assays with the stromal 600-kDa fractions in the presence of the four different nicotinamide-adenine dinucleotides indicate that PRK activity depends on complex dissociation and might be further regulated by the accessible ratio of NADP/NADPH. From these results, we conclude that light regulation of the Calvin cycle in higher plants is not only via reductive activation of different proteins by the well-established ferredoxin/thioredoxin system, but in addition, by reversible dissociation of the PRK/CP12/GAPDH complex, mediated by NADP(H).

Higher-plant chloroplast NAD(P)-glyceraldehyde 3-phosphate dehydrogenase (NAD(P)-GAPDH; EC 1.2.1.13) is composed of two different nuclear-encoded subunits, GAPA and GAPB, forming the highly active heterotetrameric A2B2 enzyme. The main difference between these two subunits is a C-terminal extension of about 30 amino acid residues of GAPB. We present cDNA clones for a nuclear-encoded chloroplast protein from pea, spinach and tobacco, which we have named CP12. The mature protein consists of only 74, 75 and 76 amino acid residues, respectively and contains two domains with significant homology to the C-terminal extension of GAPB. Affinity chromatography approaches reveal also a specific interaction between CP12 and chloroplast GAPDH. Northern blot analysis indicates that CP12 is, like plastid GAPDH, expressed in green and also in etiolated leaves. Further homology is observed between CP12 and ORF3, an open reading frame located in the hox gene cluster of Anabaena variabilis. This gene cluster encodes the subunits of the bidirectional NADP(+)-dependent [NiFeS] dehydrogenase. We propose therefore a common evolutionary origin of CP12 and higher-plant chloroplast GAPDH subunit GAPB from the cyanobacterial ORF3.

A phase I pharmacokinetic and safety clinical trial of rBPI23, a recombinant amino terminal fragment of bactericidal/permeability-increasing protein, was conducted in healthy male volunteers. rBPI23 was administered as a 5 or 30 min infusion at doses of .1 to 1 mg/kg. The pharmacokinetics of rBPI23 in human subjects were described by a bi-exponential disposition function with evidence of concentration-dependent kinetics. The alpha half-life increased significantly with increasing dose, from 4-5 min at .1 mg/kg to 7-8 min at 1 mg/kg. The beta half-life varied between 18 and 29 min regardless of dose and the clearance varied from 5 to 10 mL/min/kg. Very little, if any, of the administered rBPI23 was excreted intact in the urine. Electrocardiograms, ionized calcium concentration, prothrombin and partial prothrombin times, hematologic parameters, and blood chemistries remained normal. Furthermore, no antibody response to rBPI23 was observed in any of the subjects.

Bactericidal/permeability-increasing protein (BPI) is a neutrophil azurophilic granule component that is bactericidal towards Gram-negative bacteria and inhibits lipopolysaccharide-mediated inflammatory responses. We conducted a prospective study to measure plasma BPI concentrations in 36 critically ill children with and without the sepsis syndrome. Plasma BPI concentrations ranged from 0.5 to 452 ng/ml. Patients with the sepsis syndrome had higher median plasma BPI concentrations than critically ill controls (5.1 vs. 1.8 ng/ml, P = 0.006). Patients with organ system failure had higher median plasma BPI concentrations than those with no organ system failure (4.5 vs. 1.3 ng/ml, P = 0.001). Plasma BPI concentrations were positively associated with pediatric risk of mortality score (P = 0.03, rs = 0.4). These data provide the first clinical insights regarding the role of endogenous BPI production in critically ill children and suggest that BPI may play an important role in host defenses.

A recombinant endotoxin-neutralizing protein, rBPI23, was shown to partially prevent endotoxin-induced activation of the fibrinolytic and coagulation systems in experimental endotoxemia in humans. In a placebo-controlled, blinded crossover study, eight volunteers were challenged twice with an intravenous bolus injection of endotoxin (40 EU/kg of body weight) and concurrently received either rBPI23 (1 mg/kg) or placebo (human serum albumin, 0.2 mg/kg). rBPI23 treatment significantly lowered the endotoxin-induced fibrinolytic response, ie, reduced the release of tissue-type plasminogen activator, urokinase-type plasminogen activator, plasminogen activator inhibitor antigen, and complex formation of plasmin alpha 2-antiplasmin (P = .0078 for each). Plasminogen activator inhibitor activity was also reduced, but not significantly according to the Hochberg method (P = .0304). The endotoxin-induced activation of the procoagulant state as reflected by increase in F1 + 2 fragments and TAT complexes was blunted by rBPI23 infusion (P = .0391 [not significant according to the Hochberg method] and .0078, respectively). These results indicate that rBPI23 is capable of reducing both the activation of the fibrinolytic and the coagulation systems after low-dose endotoxin infusion in humans.

In the present study the protective effect of a recombinant endotoxin-binding protein rBPI23 on the circulatory changes in experimental endotoxemia in humans was investigated. In a controlled, blinded crossover study, eight volunteers were challenged twice with an intravenous bolus injection of endotoxin (40 EU/kg body weight), and concurrently received either rBPI23 (1 mg/kg) or placebo (human serum albumin, 0.2 mg/kg). Hemodynamic parameters were obtained non-invasively by means of M-mode, two-dimensional, and Doppler echocardiography. rBPI23 significantly reduced indices of the endotoxin-induced hyperdynamic circulation. rBPI23 treatment significantly reduced increase in cardiac index (P = 0.0156). rBPI23 treatment diminished the endotoxin-induced decrease in systemic vascular resistance index (P = 0.0304). rBPI23 did not prevent the endotoxin-induced rise in body temperature and systolic, diastolic and mean arterial pressure were not significantly different in the rBPI23- and placebo-treatment arm. Both treatment periods showed a small reduction in end diastolic and end systolic volumes. rBPI23 treatment slightly reduced the increase in M-mode ejection fraction and fractional shortening. These results indicate that rBPI23 is capable of attenuating the potentially deleterious circulatory effects of endotoxin in humans.

Recombinant phages that encode the complete precursor polypeptide for the 22 kDa polypeptide associated with photosystem II have been serologically selected from two lambda gt11 expression libraries made from polyadenylated RNA of spinach seedlings. The cDNAs hybridize to a 1.3 kb RNA species. The precursor protein is comprised of 274 amino acid residues and carries an N-terminal transit peptide of probably 69 amino acid residues. The mature protein exhibits four predicted transmembrane segments and is shown to be an integral component of photosystem II originating in a single-copy gene. The unique characteristics of this protein are: (i) it is the result of a gene-internal duplication of an ancestor with two membrane spans, (ii) a striking resemblance to LHC I/II, CP24/CP29 apoproteins, and ELIPs, although it does not bind chlorophyll and is present in cyanobacteria, and, as these proteins, (iii) it integrates into the membrane with uncleaved routing signals that display remarkable resemblance to patterns found in bipartite transit peptides.

Objective: To evaluate the safety and efficacy of E5 (Xomen-E5), a murine monoclonal immunoglobulin M antibody, in reducing mortality in patients with serious Gram-negative infections. Phase II, single-site study., Design: Randomized, double-blind, placebo-controlled study., Setting: Surgical, neurosurgical and medical ICUs, comprising approximately 30 beds in a multidisciplinary university hospital., Patients: Patients with clinical evidence of serious infection admitted to the ICU for monitoring of vital signs and for intensive care nursing. Of the 39 patients enrolled in the study, 23 had documented Gram-negative infection., Methods: Patients suspected of having life-threatening Gram-negative infections received one of two doses of E5 or placebo. Safety and efficacy were assessed by survival on days 3, 7, and 21, appearance of adverse reactions, development of antimurine antibodies, and effects on BP, urine output, WBC count, and temperature., Measurements and Main Results: Mortality rate from Gram-negative infection 3 days after the last drug (or placebo) infusion was two (22%) of nine deaths in the placebo group compared with 0 of nine for E5 2.5 mg/kg and 0 of five for E5 7.5 mg/kg. At 21 days after therapy, three patients treated with E5 had died. Only one of these three deaths resulted from infection. Eight of 15 E5 patients tested had immunoglobulin G antimurine antibodies by 3 wks after therapy began, but all were asymptomatic., Conclusions: E5 was well tolerated and may have the potential to reduce early morbidity and the mortality rate in seriously ill patients with Gram-negative infections. Results from larger phase III studies are needed to confirm these findings.

Using the expression vector lambda gt11 and immunochemical detection, six cDNA clones that encode the entire precursor polypeptides for spinach thioredoxin m were isolated and characterized. The ca. 1.0 kb cDNA sequence of the largest clone hybridizes to an RNA species of 1.1 kb. In each instance the cDNA sequences display single open reading frames encoding polypeptides of 181 amino acid residues corresponding to a molecular mass of 19.8 kDa. The sequences of the independently selected cDNAs fall into two classes that are indicative of at least two (closely related) genes for this protein. The amino acid sequences deduced from the cDNA sequences differ to some extent from the amino acid sequence published for spinach thioredoxin m. The sequences predict identical mature proteins of 112-114 amino acids corresponding to a polypeptide molecular mass of ca. 12.4-12.6 kDa, and include stroma-targeting N-terminal transit peptides of 67 residues which are removed during or after import into the organelle. Precursor protein was made in vitro from each of the different cDNA clones and imported into isolated intact chloroplasts. Independent of the cDNA clone used, two isoforms were detected in the chloroplasts after import in each instance. They comigrated with authentic thioredoxin mb and mc. These results indicate that the size variants observed for this protein in vivo result from post-translational modification and do not originate in different genes.

To determine the maximally tolerated dose of a ricin A chain-conjugated antimelanoma antibody (XomaZyme-Mel), 20 patients with metastatic melanoma were treated with escalating doses of the murine immunotoxin given as single intravenous infusion over 30 minutes. The starting dose was 0.6 mg/kg and was escalated in five groups to a maximum of 1.6 mg/kg. The maximally tolerated dose was 1.25 mg/kg as three of six patients treated at 1.6 mg/kg developed unacceptable toxicity. The dose-limiting toxicity consisted of profound fatigue, myalgias, and arthralgias. These occurred within 4 days and resolved in 7 to 10 days. Other non-dose-limiting toxicities encountered consisted of hypoalbuminemia, weight gain, peripheral edema, mild hypotension, and flu-like syndrome; the severity of these was also dose related. In addition, two allergic reactions occurred, one severe. There was one durable complete response of 12+ months' duration and one brief mixed response lasting 3 months. We conclude that the maximum tolerated single dose of XomaZyme-Mel is 1.25 mg/kg. Phase I studies evaluating 1.25 mg/kg given in multiple doses at 2- to 4-week intervals and phase II studies to determine the response rate of a single 1.25 mg/kg dose are warranted.

Xomen-E5 (E5) is a murine monoclonal IgM antibody (MAb) that binds to the lipid A epitope of endotoxin. The MAb was developed by immunization against the J5 mutant of Escherichia coli. Prior studies in humans have shown safety and T1/2 of 18.4 hours. In this double blind study patients suspected to have life threatening gram-negative infections were randomized to receive 2 doses, 24 hours apart, of placebo (P), 2.5 mg/kg E5, or 7.5 mg/kg E5. Overall 23 patients had a documented serious gram-negative infection and received at least one dose of study drug. Mortality 3 days after last infusion was 2 of 9 for P, 0 of 9 for 2.5 mg/kg, and 0 of 5 for 7.5 mg/kg. By 21 days after therapy one E5 treated patient had died. Wheezes occurred in one E5 treated patient. Eight of 15 E5 patients treated had IgG anti-murine antibodies by 3 weeks after therapy. These data suggest the need to pursue studies designed to verify that E5 reduced mortality and morbidity in seriously ill patients with gram-negative infections.

A positive effect on survival of renal grafts of pretransplant blood transfusions have been reported from several centers. The aim of this study was to study if the described graft-protecting effect of blood transfusion was present in the Gothenburg material of transplanted patients, and if this effect could be achieved by deliberately transfusing previously non-transfused patients with two units of leukocyte-reduced blood. The effect on graft survival (GS) of the number and timing of transfusions to recipients, transfusions to the cadaveric donors, HLA-A, B matching, lymphocytotoxic antibodies and pretransplant hemodialysis was also studied. The study includes 844 recipients of primary renal grafts from living related and cadaveric donors (LRD, CD) and 70 patients waiting for transplantation. In the retrospective part of the study the GS of previously transfused and non-transfused non-transfused patients was compared. In the prospective part of the study a protocol with two deliberate transfusions (DT) to previously non-transfused patients was introduced. The GS of the DT group was compared to that for patients transfused for strictly medical reasons (MT) and non-transfused patients (NT). Survival of patients and grafts was calculated according to the life table method. In the retrospective part of study one year GS in LRD transplantation was 86.6% for transfused and 38.4% for non-transfused patients (P less than 0.01). In the first period one year GS in CD transplantation was 62.1% for transfused and 35.1% for non-transfused patients (P less than 0.01). The corresponding figures in the second period were 68.1% and 39.5%, respectively (P less than 0.001). In transfused recipients receiving kidneys from transfused and non-transfused cadaveric donors, the GS was 76.3% and 55.4%, respectively (P less than 0.05). In the prospective part of study the one year GS after LRD transplantation was 85.0% in both the DT and MT groups. In CD transplantation the one year GS was 73.4% and 75.7% of the DT and MT groups, respectively. The GS of each of these two groups was significantly better than that of 20.8% for the NT group (P less than 0.01). Lymphocytotoxic antibodies were detected in 5.0% of the DT group and 23.0% of the MT group (P less than 0.001). Foreign HLA-B series antigens had a negative influence on GS in the first period of the retrospective CD study. Later, no influence on GS was noted of HLA-A, B matching. Hemodialysis prior to transplantation did not influence GS.(ABSTRACT TRUNCATED AT 400 WORDS)

A method for studying the ultrastructure of cells grown on hydroxyethylmethacrylate (HEMA) hydrogels is described. Under normal conditions, HEMA hydrogels tend to swell when placed in hypotonic solutions and to shrink during alcohol dehydration. To overcome this severe swelling and/or shrinking, all solutions used during the fixation procedure are made in Puck's saline G, and dehydration is accomplished with a graded series of ethanol solutions prepared with Puck's saline G and polyethylene glycol. Infiltration of the sample with embedding material is achieved with the aid of a vacuum oven. Our findings suggest that the cells cultured on collagen hydrogels are ultrastructurally indistinguishable from those cultured on tissue-culture plastic. In addition, the unusual crater-like topography of the hydrogel can be utilized as an experimental aid in the study of cell attachment and spreading.

The mechanism by which Clostridium difficile toxin B causes cells in culture to round was investigated. Cultured human lung fibroblasts and rabbit aortic smooth muscle cells were treated with partially purified or purified toxin B and monitored by light and transmission electron microscopy (TEM). Both preparations caused progressive cell rounding which correlated with disorganization of actin-containing myofilament bundles. Thin myofilaments became fragmented and finally disappeared (after 24 h) and dense bodies became more prominent, while all other organelles appeared unaffected.