Variation of the detergent-binding capacity and phospholipid content of membrane proteins when purified in different detergents.

Purified membrane proteins are ternary complexes consisting of protein, lipid, and detergent. Information about the amounts of detergent and endogenous phospholipid molecules bound to purified membrane proteins is largely lacking. In this systematic study, three model membrane proteins of different oligomeric states were purified in nine different detergents at commonly used concentrations and characterized biochemically and biophysically. Detergent-binding capacities and phospholipid contents of the model proteins were determined and compared. The insights on ternary complexes obtained from the experimental results, when put into a general context, are summarized as follows. 1), The amount of detergent and 2) the amount of endogenous phospholipids bound to purified membrane proteins are dependent on the size of the hydrophobic lipid-accessible protein surface areas and the physicochemical properties of the detergents used. 3), The size of the detergent and lipid belt surrounding the hydrophobic lipid-accessible surface of purified membrane proteins can be tuned by the appropriate choice of detergent. 4), The detergents n-nonyl-β-D-glucopyranoside and Cymal-5 have exceptional delipidating effects on ternary complexes. 5), The types of endogenous phospholipids bound to membrane proteins can vary depending on the detergent used for solubilization and purification. 6), Furthermore, we demonstrate that size-exclusion chromatography can be a suitable method for estimating the molecular mass of ternary complexes. The findings presented suggest a strategy to control and tune the numbers of detergent and endogenous phospholipid molecules bound to membrane proteins. These two parameters are potentially important for the successul crystallization of membrane proteins for structure determination by crystallographic approaches.

Elucidating the Functional Relationship Between Working Memory Capacity and Psychometric Intelligence: A Fixed-Links Modeling Approach for Experimental Repeated-Measures Designs

Numerous studies reported a strong link between working memory capacity (WMC) and fluid intelligence (Gf), although views differ in respect to how close these two constructs are related to each other. In the present study, we used a WMC task with five levels of task demands to assess the relationship between WMC and Gf by means of a new methodological approach referred to as fixed-links modeling. Fixed-links models belong to the family of confirmatory factor analysis (CFA) and are of particular interest for experimental, repeated-measures designs. With this technique, processes systematically varying across task conditions can be disentangled from processes unaffected by the experimental manipulation. Proceeding from the assumption that experimental manipulation in a WMC task leads to increasing demands on WMC, the processes systematically varying across task conditions can be assumed to be WMC-specific. Processes not varying across task conditions, on the other hand, are probably independent of WMC. Fixed-links models allow for representing these two kinds of processes by two independent latent variables. In contrast to traditional CFA where a common latent variable is derived from the different task conditions, fixed-links models facilitate a more precise or purified representation of the WMC-related processes of interest. By using fixed-links modeling to analyze data of 200 participants, we identified a non-experimental latent variable, representing processes that remained constant irrespective of the WMC task conditions, and an experimental latent variable which reflected processes that varied as a function of experimental manipulation. This latter variable represents the increasing demands on WMC and, hence, was considered a purified measure of WMC controlled for the constant processes. Fixed-links modeling showed that both the purified measure of WMC (β = .48) as well as the constant processes involved in the task (β = .45) were related to Gf. Taken together, these two latent variables explained the same portion of variance of Gf as a single latent variable obtained by traditional CFA (β = .65) indicating that traditional CFA causes an overestimation of the effective relationship between WMC and Gf. Thus, fixed-links modeling provides a feasible method for a more valid investigation of the functional relationship between specific constructs.

Forest regeneration capacity and its enhancement by forest management and silviculture in Cabo Delgado

SDC has been involved in rural development in Cabo Delgado for more than 30 years. Shortly after the independence of Mozambique, projects in water supply and integrated rural development were initiated. The silvoagropastoral project FO9 based in Mueda was a very early experience in forestry in Cabo Delgado. Andreas Kläy was responsible for the forestry sector in FO9 for 3 years in the early 1980s and had an opportunity to initiate an exchange of ideas and experience in rural development theory and approaches with Yussuf Adam, who was doing research in human anthropology and history in the province. 25 years later, the current situation of forest management in Cabo Delgado was reassessed, with a specific focus on concessions in the North. The opportunity for a partnership between the MITI SA, the University of Eduardo Mondlane, and CDE was created on the basis of this preliminary study1. The aim of this partnership is to generate knowledge and develop capacity for sustainable forest management. The preliminary study showed that “…we have to face weaknesses and would like to start a learning process with the main institutions, organisations, and stakeholder groups active in forest management and research in the North of Cabo Delgado. This learning process will involve studies supported by competent research institutions and workshops …” The specific objectives of ESAPP project Q804 are the following: 1. Contribute to understanding of the forestry sector; 2. Capacity development for professionals and academics; 3. Support for the private sector and the local forest service; 4. Support data generation at Cabo Delgado's Provincial Service; 5. Capacity development for Swiss academic institutions (CDE and ETHZ). A conceptual planning platform was elaborated as a basis for cooperation and research in the partnership (cf. Annex 1). The partners agreed to work on two lines of research: biophysical and socio-economic. In order to ensure a transdisciplinary approach, disciplinary research is anchored in common understanding in workshops based on the LforS methods. These workshops integrate the main stakeholders in the local context of the COMADEL concession in Nangade District managed by MITI SA, and take place in the village of Namiune. The research team observed that current management schemes consist mainly of strategies of nature mining by most stakeholders involved. Institutional settings - formal and informal - have little impact due to weak capacity at the local level and corruption. Local difficulties in a remote rural area facilitate external access to resources and are perpetuated by the loss of benefits. The benefits of logging remain at the top level (economic and political elites). The interests of the owners of the concession in stopping the loss of resources caused by this regime offers a unique opportunity to intervene in the logic of resource degradation and agony in rural development and forest management.

PreImplantation Factor bolsters neuroprotection via modulating Protein Kinase A and Protein Kinase C signaling

A synthetic peptide (sPIF) analogous to the mammalian embryo-derived PreImplantation Factor (PIF) enables neuroprotection in rodent models of experimental autoimmune encephalomyelitis and perinatal brain injury. The protective effects have been attributed, in part, to sPIF's ability to inhibit the biogenesis of microRNA let-7, which is released from injured cells during central nervous system (CNS) damage and induces neuronal death. Here, we uncover another novel mechanism of sPIF-mediated neuroprotection. Using a clinically relevant rat newborn brain injury model, we demonstrate that sPIF, when subcutaneously administrated, is able to reduce cell death, reverse neuronal loss and restore proper cortical architecture. We show, both in vivo and in vitro, that sPIF activates cyclic AMP dependent protein kinase (PKA) and calcium-dependent protein kinase (PKC) signaling, leading to increased phosphorylation of major neuroprotective substrates GAP-43, BAD and CREB. Phosphorylated CREB in turn facilitates expression of Gap43, Bdnf and Bcl2 known to have important roles in regulating neuronal growth, survival and remodeling. As is the case in sPIF-mediated let-7 repression, we provide evidence that sPIF-mediated PKA/PKC activation is dependent on TLR4 expression. Thus, we propose that sPIF imparts neuroprotection via multiple mechanisms at multiple levels downstream of TLR4. Given the recent FDA fast-track approval of sPIF for clinical trials, its potential clinical application for treating other CNS diseases can be envisioned.

The characterization of mouse carboxypeptidase N small subunit gene structure and presence in developing embryos

Carboxypeptidase N (CPN) is a plasma zinc metalloprotease, which consists of two enzymatically active small subunits and two large subunits that protect the protein from degradation. CPN cleaves carboxy-terminal arginines and lysines from peptides found in the bloodstream such as complement anaphylatoxins, kinins, and creatine kinase MM. In this study, the mouse CPN small subunit (CPN1) coding region, gene structure, and chromosomal location were characterized and the expression of CPN1 was investigated in mouse embryos at different stages of development. The CPN1 gene, which was approximately 29 kb in length, contained nine exons and localized to mouse chromosome 19D2. The fifth and sixth exons of CPN1 encoded the amino acids necessary for substrate binding and catalytic activity. CPN1 RNA was expressed predominately in adult liver and contained a 1371 bp open reading frame encoding 457 amino acids. In the mouse embryo, CPN1 RNA was observed at 8.5 days post coitus (dpc), while its protein was detected at 10.5 dpc. In situ hybridization of the fetal liver detected CPN1 RNA in erythroid progenitor cells at 10.5, 13.5, and 16.5 dpc and in hepatocytes at 16.5 dpc. This was compared to the expression of the complement component C3, the parent molecule of complement anaphylatoxin C3a. Consistently throughout the experiments, CPN1 message and protein preceded the expression of C3. To obtain a better understanding of the biological significance of CPN1 in vivo, studies were initiated to produce a genetically engineered mouse in which the CPN1 gene was ablated. To facilitate this project a targeting vector was constructed by removing the functionally important fifth and sixth exons of the CPN1 gene. Collectively, these studies have: (1) provided important detailed information regarding the structure and organization of the murine CPN1 gene, (2) yielded insights into the developmental expression of mouse CPN1 in relationship to C3 expression, and (3) set the stage for the generation of a CPN1 “knock-out” mouse, which can be used to determine the biological significance of CPN1 in both normal and diseased conditions. ^

Bexar County's dental safety net for children: An estimate of our current capacity and need

Introduction. It has been well established that poor uninsured children lack access to dental care and have greater dental needs than their insured counterparts. ^ Objective. To assess the capacity of Bexar County's dental safety net to treat children. To assess the dental needs of Bexar County children ages 0-18 who are uninsured or are Medicaid or SCHIP recipients. ^ Methods. Information was requested from dental safety net clinics that treat children ages 0-18. Data from the census, NHANES and other sources was used to estimate the dental needs. ^ Results. The capacity of the current safety net to treat children is 33,537 patient encounters per year. The dental needs of the community are 227,124 patient encounters per year. ^ Conclusion. The results of the study indicate that Bexar County is not prepared to treat the dental needs of the underserved children in San Antonio.^

Trauma care capacity and performance in Texas: Regional comparisons

Objective. The study reviewed one year of Texas hospital discharge data and Trauma Registry data for the 22 trauma services regions in Texas to identify regional variations in capacity, process of care and clinical outcomes for trauma patients, and analyze the statistical associations among capacity, process of care, and outcomes. ^ Methods. Cross sectional study design covering one year of state-wide Texas data. Indicators of trauma capacity, trauma care processes, and clinical outcomes were defined and data were collected on each indicator. Descriptive analyses were conducted of regional variations in trauma capacity, process of care, and clinical outcomes at all trauma centers, at Level I and II trauma centers and at Level III and IV trauma centers. Multilevel regression models were performed to test the relations among trauma capacity, process of care, and outcome measures at all trauma centers, at Level I and II trauma centers and at Level III and IV trauma centers while controlling for confounders such as age, gender, race/ethnicity, injury severity, level of trauma centers and urbanization. ^ Results. Significant regional variation was found among the 22 trauma services regions across Texas in trauma capacity, process of care, and clinical outcomes. The regional trauma bed rate, the average staffed bed per 100,000 varied significantly by trauma service region. Pre-hospital trauma care processes were significantly variable by region---EMS time, transfer time, and triage. Clinical outcomes including mortality, hospital and intensive care unit length of stay, and hospital charges also varied significantly by region. In multilevel regression analysis, the average trauma bed rate was significantly related to trauma care processes including ambulance delivery time, transfer time, and triage after controlling for age, gender, race/ethnicity, injury severity, level of trauma centers, and urbanization at all trauma centers. Transfer time only among processes of care was significant with the average trauma bed rate by region at Level III and IV. Also trauma mortality only among outcomes measures was significantly associated with the average trauma bed rate by region at all trauma centers. Hospital charges only among outcomes measures were statistically related to trauma bed rate at Level I and II trauma centers. The effect of confounders on processes and outcomes such as age, gender, race/ethnicity, injury severity, and urbanization was found significantly variable by level of trauma centers. ^ Conclusions. Regional variation in trauma capacity, process, and outcomes in Texas was extensive. Trauma capacity, age, gender, race/ethnicity, injury severity, level of trauma centers and urbanization were significantly associated with trauma process and clinical outcomes depending on level of trauma centers. ^ Key words: regionalized trauma systems, trauma capacity, pre-hospital trauma care, process, trauma outcomes, trauma performance, evaluation measures, regional variations ^

Evidence that lymphokine -activated killer (LAK) cell function is regulated by both serine and tyrosine kinase pathways

Signal transduction pathways operative in lymphokine activated killer (LAK) cells during execution of cytolytic function have never been characterized. Based on ubiquitous involvement of protein phosphorylation in activation of cytolytic mechanisms used by CTL and NK cells, it was hypothesized that changes in protein phosphorylation should occur when LAK encounter tumor targets. It was further hypothesized that protein kinases would regulate LAK-mediated cytotoxicity. Exposure to either SK-Mel-1 (melanoma) or Raji (lymphoma) targets consistently led to increased phosphorylation of two 65-kD LAK proteins pp65a and -b, with isoelectric points (pI) of 5.1 and 5.2 respectively. Increased p65 phosphorylation was initiated between 1 and 5 min after tumor coincubation, occurred on Ser residues, required physical contact between LAK and tumors, correlated with target recognition, and also occurred after crosslinking Fc$\gamma$RIIIA in the absence of tumors. Both pp65a and -b were tentatively identified as phosphorylated forms of the actin-bundling protein L-plastin, based on pI, molecular weight, and cross-reactivity with specific antiserum. The known biochemical properties of L-plastin suggest it may be involved in regulating adhesion of LAK to tumor targets. The protein tyrosine kinase-specific inhibitor Herb A did not block p65 phosphorylation, but blocked LAK killing of multiple tumor targets at a post-binding stage. Greater than 50% inhibition of cytotoxicity was observed after a 2.5-h pretreatment with 0.125 $\mu$g/ml Herb A. Inhibition occurred over a period in pretreatment which LAK were not dependent upon IL-2 for maintenance of killing activity, supporting the conclusion that the drug interfered with mobilization of cytotoxic function. Granule exocytosis measured by BLT-esterase release from LAK occurred after coincubation with tumors, and was inhibited by Herb A LAK cytotoxicity was dependent upon extracellular calcium, suggesting that granule exocytosis rather than Fas ligand was the principal pathway leading to target cell death. The data indicate that protein tyrosine kinases play a pivotal role in LAK cytolytic function by regulating granule exocytosis, and that tumor targets can activate an adhesion dependent Ser kinase pathway in LAK resulting in phosphorylation of L-plastin. ^

(Table 1) Cation exchange capacity and other characteristics of soils common in Lower Saxony, Germany

The chemical and biochemical processes associated with the filtration of rainwater through soils, a step in groundwater recharge, were investigated. Under simulated climatic conditions in the laboratory, undisturbed soil columns of partly loamy sands, sandy soils and loess were run as lysimeters. A series of extraction procedures was carried out to determine solid matter in unaltered rock materials and in soil horizons. Drainage water and moisture movement in the columns were analysed and traced respectively. The behaviour of soluble humic substance was investigated by percolation and suspension experiments. The development of seepage-water in the unsaturated zone is closely associated with the soil genetic processes. Determining autonomous chemical and physical parameters are mineral composition and grain size distribution in the original unconsolidated host rock and prevailing climatic conditions. They influence biological activity and transport of solids, dissolved matter and gases in the unsaturated zone. Humic substances, either as amorphous solid matter or as soluble humic acids play a part in diverse sorption, solution and precipitation processes.

Exchange capacity and chemical composition of Fe-Mn nodules from the Central Pacific

Constants of calcium, magnesium, zinc, cobalt, copper, and nickel exchange for sodium in iron-manganese nodules taken from different parts of the Pacific Ocean were determined under static conditions at constant ionic strength (?=0.05). These determinations revealed high capacity of nodules for sorbing the referred ions (their exchange constants range from 1.93 to 20.85). Obtained data demonstrate the major role of MnO and Fe2O3 in sorption processes in iron-manganese nodules.

Cation exchange capacity and water content of ODP sites from Nankai

Pore fluid chlorinity lower than seawater is often observed in accretionary wedges and one of the possible causes of pore water freshening is the smectite to illite reaction. This reaction occurs during diagenesis in the 80-150°C temperature range. Low chlorinity anomalies observed at the toe of accretionary wedges have thus been interpreted as evidence for lateral fluid migration from inner parts of the wedge and the seismogenic zone. However, temperature conditions in Nankai Trough are locally high enough for the smectite to illite transition to occur in situ. Cation exchange capacity is here used as a proxy for smectite content in the sediment and the amount of interlayer water released during the smectite to illite reaction represents in average 12 water molecules per cation charge. Water and chloride budget calculations show that there is enough smectite to explain the chlorinity anomalies by in situ reactions. The shape of the pore fluid chlorinity profiles can be explained if compaction is also taken into account in the model. Lateral flow is not needed. This argument, based solely on chloride concentration, does not imply that lateral flow is absent. However, previous estimations of lateral fluid fluxes, and of the duration of transient flow events along the de.collement, should be reconsidered.