Interaction between drug delivery vehicles and cells under the effect of shear stress.

Over the last decades, researchers have developed an ever greater and more ingenious variety of drug delivery vehicles (DDVs). This has made it possible to encapsulate a wide selection of therapeutic agents, ranging from proteins, enzymes, and peptides to hydrophilic and hydrophobic small drugs while, at the same time, allowing for drug release to be triggered through a diverse range of physical and chemical cues. While these advances are impressive, the field has been lacking behind in translating these systems into the clinic, mainly due to low predictability of in vitro and rodent in vivo models. An important factor within the complex and dynamic human in vivo environment is the shear flow observed within our circulatory system and many other tissues. Within this review, recent advances to leverage microfluidic devices to better mimic these conditions through novel in vitro assays are summarized. By grouping the discussion in three prominent classes of DDVs (lipidic and polymeric particles as well as inorganic nanoparticles), we hope to guide researchers within drug delivery into this exciting field and advance a further implementation of these assay systems within the development of DDVs.

Swedish translation and validation of a web-based questionnaire for registration of overuse problems.

The main aim of this study was to translate the Oslo Sport Trauma Research Center (OSTRC) Overuse Injury Questionnaire into Swedish. The validity and applicability of the questionnaire for studying overuse injuries among Swedish handball, volleyball, tennis, and orienteering top athletes were also examined. The back-translation method was used for translation. An expert committee further developed it for use in a study of injuries in handball, orienteering, tennis, and volleyball. A 10-week pretest was then conducted on 43 athletes, average age 21 (18-31) from these sports, during which time the athletes completed the modified OSTRC questionnaire on a weekly basis. In the 10th week, four additional questions were added in order to examine the questionnaire's content validity. No major disagreement was found in the translation. The athletes perceived the web-based questionnaire to be smooth and easy to complete, accurately capturing overuse injuries. However, suggestions were made to add questions relating to the hip for orienteerers and to the hand/fingers for handball players. The average prevalence of overuse injuries for all athletes, in any anatomical area was 22% (95% confidence interval 20-25). Construct validity appeared to be high, and we therefore suggest that the questionnaire may be used when studying overuse injuries in different sports.

Perceptions and care seeking behavior of obstetric complication in Thailand.

BACKGROUND: Importance of maternal health has been recognized over the last decade, however information about the perception of illness and healthcare behavior of obstetric complication is lacking. OBJECTIVE: This study assesses women ' s knowledge, perception, and experience of obstetric complication and care-seeking behavior and explores the factors associated with the morbidity and the constraints hindering them from seeking timely care. METHODS: Twenty one in-depth interviews on the perceptions, experience and care seeking behavior related to pregnancy and delivery of Women at Kanchanaburi Demographic Surveillance site of Thailand were conducted. A structured guideline was first prepared in English and translated into Thai language. An interpreter was hired to interview women at the Thai-Myanmar border to translate Thai into local language. A moderator note-taker, and interpreter were present throughout the interview period and tape recorded the conversation. RESULTS: In-depth interview revealed that even though quality maternal health care was accessible to most of the women, obstetric complication was prevalent and they were not seeking appropriate care specifically in highland. Too early and too late marriage, frequent child bearing, poverty, hard work, poor nutrition and traditional practices were the reasons for complications. Poor transportation, lack of health insurance, inadequate training of health personnel, poor health facilities and the perception that the complications are normal for pregnant women were the main reasons for not seeking appropriate care. CONCLUSIONS: Perceived reasons for complications among women living in Kanchanaburi, Thailand were early marriage, frequent childbearing, hard work, poor nutrition and traditional practices. The constraints hindering them from seeking care for the complications were perceived to be the lack of access to health personnel, health facilities, and proper transportation. These issues seemed to be related to poverty.

Selected quality characteristics of fresh-cut sweet potatoes coated with chitosan during 17-day refrigerated storage.

Selected quality characteristics of fresh-cut sweet potatoes (FCSP) coated with chitosan were evaluated during 17-d refrigerated storage. The FCSP cubes were coated with a solution (1%, w/v) of chitosan having 470 or 1110 kDa. Color (L*, a*, b*) values of uncoated and chitosan-coated FCSP during storage were generally affected by storage time as well as coating treatments (P < 0.05). No significant changes in color lightness (L*) of 470 kDa-coated FCSP were observed during the 17-d storage. During days 3 to 17, 470 kDa-coated FCSP had significantly higher redness (a*) and yellowness (b*) values than did uncoated and 1110 kDa-coated FCSP. Texture firmness of uncoated and chitosan-coated FCSP exhibited minimal changes during the 17-d storage. Although actual weight loss values (%) of uncoated and chitosan-coated FCSP were not significantly different at day 17, the weight loss difference (%) between day 3 and day 17 for uncoated FCSP (3.02%) was slightly higher compared to those (2.24% to 2.26%) of chitosan-coated FCSP. The initial total aerobic count was 4.7 log(10) CFU/g which then gradually increased to 8.54 and 9.67 log(10) CFU/g after 17 d of storage for 470 kDa-coated and uncoated FCSP, respectively. After day 6, the total aerobic counts of uncoated FCSP were higher than those of 470 kDa-coated FCSP. The yeast and mold count of chitosan-coated FCSP was about 2.5 log(10) CFU/g at day 17. Overall, consumers could not differentiate between 470 kDa-coated FCSP at day 17 and uncoated FCSP at day 0.

Quantum chaotic environments, the butterfly effect, and decoherence.

We investigate the sensitivity of quantum systems that are chaotic in a classical limit to small perturbations of their equations of motion. This sensitivity, originally studied in the context of defining quantum chaos, is relevant to decoherence when the environment has a chaotic classical counterpart.

Phosphorylation of human plasma alpha2-Heremans-Schmid glycoprotein (human fetuin) in vivo.

A fraction of alpha2-Heremans-Schmid (alpha2-HS) glycoprotein (human fetuin) isolated from plasma was phosphorylated at serine-120 and serine-312 as shown by MS and peptide fragment sequencing after tryptic digestion. Serine-312-containing peptides were phosphorylated to 77% as determined from relative peak heights in the mass spectrum, which together with the phosphorylation of serine-120 implies a molar degree of phosphorylation of at least 1. Approximately 20% of the circulating fetuin plasma pool was phosphorylated to approx. 1 mol of phosphate/mol of protein. The remainder did not contain phosphate, resulting in an average phosphorylation degree for the protein in plasma of approx. 0.2 mol/mol. The isolated alpha2-HS glycoprotein was a heterodimer in which the entire C-terminal part of the connecting peptide including threonine-321 was present, but traces of C-terminally trimmed connecting peptide fragments were also found. The short B-chain was O-glycosylated to approx. 40%, whereas the N-glycosylation of asparagine-138 and asparagine-158 seemed to be 100%. This finding, for the first time, that circulating human plasma fetuin is partly phosphorylated, implies that the effects of phosphorylated alpha2-HS glycoprotein on insulin signal transduction seen in different cell systems could be relevant to its physiological function in vivo.

Bi-substrate analogue ligands for affinity chromatography of protein kinases.

Novel affinity ligands, consisting of ATP-resembling part coupled with specificity determining peptide fragment, were proposed for purification of protein kinases. Following this approach affinity sorbents based on two closely similar ligands AdoC-Aoc-Arg4-Lys and AdoC-Aoc-Arg4-NH(CH2)6NH2, where AdoC stands for adenosine-5'-carboxylic acid and Aoc for amino-octanoic acid, were synthesized and tested for purification of recombinant protein kinase A catalytic subunit directly from crude cell extract. Elution of the enzyme with MgATP as well as L-arginine yielded homogeneous protein kinase A preparation in a single purification step. Also protein kinase A from pig heart homogenate was selectively isolated using MgATP as eluting agent. Protein kinase with acidic specificity determinant (CK2) as well as other proteins possessing nucleotide binding site (L-type pyruvate kinase) or sites for wide variety of different ligands (bovine serum albumin) did not bind to the column, pointing to high selectivity of the bi-functional binding mode of the affinity ligand.

Calcium-dependent protein kinase from maize seedlings activated by phospholipids.

A calcium- and phospholipid-dependent protein kinase of apparent molecular mass 54 kDa (designated ZmCPKp54) was partially purified from etiolated maize seedlings. Activity of ZmCPKp54 is stimulated by phosphatidylserine and phosphatidylinositol, but is not essentially affected by diolein and phorbol esters. The enzyme cross-reacts with polyclonal antibodies against the calmodulin like-domain of the calcium-dependent protein kinase, but not with antibodies against catalytic or regulatory domains of protein kinase C. ZmCPKp54 is not able to phosphorylate the specific substrates of protein kinase C (MARCKS peptide and protein kinase C substrate peptide derived from pseudosubstrate sequence) and its activity is not inhibited by specific PKC inhibitors (bisindolylmaleimide, protein kinase C pseudosubstrate inhibitory peptide). The substrate specificity and sensitivity to the inhibitors of the maize enzyme resembles calcium-dependent protein kinase. The biochemical and immunological properties indicate that ZmCPKp54 belongs to the calcium-dependent protein kinase family.

Alteration of the fibrinogen molecule and its phosphorylation state in myocardial infarction patients undergoing thrombolytic treatment.

Fibrinogen was purified by protamine-agarose chromatography from plasma from three patients after their submission to hospital due to acute myocardial infarction. The total amount of phosphate bound to fibrinogen and the concentration of fibrinogen was determined in samples withdrawn immediately after submission and after thrombolytic treatment. Streptokinase treatment almost totally removed circulating fibrinogen while recombinant tissue plasminogen activator spared much of it. In patients treated with streptokinase, the new circulating fibrinogen was homogeneous according to the single alpha-band seen after sodium dodecyl sulphate polyacrylamide gel electrophoresis analysis under reducing conditions, whereas fibrinogen from the recombinant tissue plasminogen activator-treated patient as well as healthy controls exhibited two alpha-bands in the 66-kDa region. The molar ratios of phosphate to fibrinogen of healthy controls and commercial fibrinogen were 0.82 (+/-0.04) and 0. 87 (+/-0.05), respectively. For two streptokinase-treated patients the degree of phosphorylation increased threefold from a normal range of 0.97 (+/-0.11) and 0.67 (+/-0.09) mol/mol fibrinogen before treatment to 3.33 (+/-0.32) and 1.86 (+/-0.17) mol/mol in newly formed fibrinogen on day 1. Recombinant tissue plasminogen activator treatment led to a smaller increase in phosphorylation, from 1.14 (+/-0.13) pretreatment to 1.65 (+/-0.11) after treatment on day 1. In conclusion we show in this report that after streptokinase treatment of patients with acute myocardial infarction, the new Aalpha-chain of fibrinogen was a homogeneous single 66-kDa band on sodium dodecyl sulphate polyacrylamide gel electrophoresis under reducing conditions and that the degree of phosphorylation of plasma fibrinogen was elevated, approaching the theoretical limit of 4 mol phosphate/mol fibrinogen.

Peptide phosphorylation by calcium-dependent protein kinase from maize seedlings.

Ca2+-dependent protein kinase (CDPK-1) was purified from maize seedlings, and its substrate specificity studied using a set of synthetic peptides derived from the phosphorylatable sequence RVLSRLHS15VRER of maize sucrose synthase 2. The decapeptide LARLHSVRER was found to be efficiently phosphorylated as a minimal substrate. The same set of peptides were found to be phosphorylated by mammalian protein kinase Cbeta (PKC), but showed low reactivity with protein kinase A (PKA). Proceeding from the sequence LARLHSVRER, a series of cellulose-membrane-attached peptides of systematically modified structure was synthesised. These peptides had hydrophobic (Ala, Leu) and ionic (Arg, Glu) amino acids substituted in each position. The phosphorylation of these substrates by CDPK-1 was measured and the substrate specificity of the maize protein kinase characterised by the consensus sequence motif A/L-5X-4R-3X-2X-1SX+1R+2Z+3R+4, where X denotes a position with no strict amino acid requirements and Z a position strictly not tolerating arginine compared with the other three varied amino acids. This motif had a characteristic sequence element RZR at positions +2 to +4 and closely resembled the primary structure of the sucrose synthase phosphorylation site. The sequence surrounding the phosphorylatable serine in this consensus motif was similar to the analogous sequence K/RXXS/TXK/R proposed for mammalian PKC, but different from the consensus motif RRXS/TX for PKA.

Kinetic study of the inhibition of CK2 by heparin fragments of different length.

The structure-activity relationships for the inhibition of protein kinase CK2 by heparin were investigated using purified heparin fragments of different length, varying from 4 to 24 oligosaccharide sugar units. The inhibitory potency was shown to decrease concomitant with the shortening of the heparin fragment length. The fragment of 24 oligosaccharide sugar units was the most potent inhibitor with a K(i) value of 22 nM which is close to the K(i) value for the commercial heparin mixture available. Shortening of the heparin from 24 to 12 sugar units had a moderate influence on the inhibitory potency causing an increase in K(i) values up to 151 nM while fragments shorter than 12 sugar units showed a more drastic increase in K(i) values reaching up to micromolar range. The mode of inhibition was studied in respect to the protein substrate beta-casein and it was shown to be competitive for the long as well as for the short heparin fragments. In contrast, the inhibition mode in respect to a synthetic peptide substrate RRRADDSDDDDD was found to be hyperbolic partial non-competitive mixed-type. Such a kinetic model suggests that heparin binds to a site on CK2 which does not overlap with the peptide substrate binding site and that a productive enzyme complex exists where both heparin and peptide substrate are simultaneously bound. This is in contrast to the competitive inhibition model of the phosphorylation of protein substrate beta-casein where the binding of the protein substrate and inhibitor was mutually exclusive.

Adenosine-5'-carboxylic acid peptidyl derivatives as inhibitors of protein kinases.

A new class of protein kinase bisubstrate-analog inhibitors was designed on the basis of adenosine-5'-carboxylic acid derivatives, where a short peptide was attached to the 5'-carbon atom of the adenosine sugar moiety via a linker chain. The potency and selectivity of these inhibitors were adjusted by relevant combination of these structural fragments, resembling the structure of the bisubstrate complex of the peptide phosphorylation reaction.

KDEL motif interacts with a specific sequence in mammalian erd2 receptor.

The ER retention of lumenal proteins is achieved by a process which involves binding of escaped proteins via the C-terminal KDEL-tags to a KDEL receptor (erd2 receptor) in a post-ER compartment and return of the protein-receptor complex back to the ER. The transmembrane topology of the human KDEL receptor, which is an integral membrane protein, has been proposed. We have synthesised sets of cellulose-bound overlapping peptides covering the complete se quence of the receptor to study the interaction of the erd2 receptor with lumenal ER proteins, CaBP1 and CaBP2. At the next stage, the proposed lumenal loops of the receptor were more closely mapped. A short sequence, essential for the protein binding to the most efficient binding site of the receptor, was identified as 22KIWK25, which is in accordance with one of the proposed structural models of the receptor. The binding was of high specificity and was almost completely inhibited by KDEL-containing soluble peptides. The phosphorylation state of CaBP1/CaBP2 did not affect their binding to the KDEL receptor.

Erythrocytic protein kinase C activity in primary hypertension.

OBJECTIVES: Increased protein kinase C activity has been reported in erythrocytes from patients with primary hypertension and also from hypertensive rats. In this phenomenological study, we investigated whether a possible increased activity was the result of an augmented amount of enzyme molecules or a more active enzyme. DESIGN: Collect blood samples, separate erythrocytes from other blood cells. After partial purification of protein kinase C in the erythrocyte lysate, assay the enzyme activity under optimal conditions using a specific peptide substrate. SETTING: Central Hospital in Eskilstuna and University Hospital in Uppsala, Sweden. SUBJECTS: Healthy individuals: 47 persons (20 women and 27 men). Ten patients with untreated primary hypertension. MAIN OUTCOME MEASURES: Erythrocytes were separated from leucocytes and platelets by passing through a cellulose column followed by repeated washings. Some proteins in the erythrocyte lysate interfering with protein kinase C assay were removed by chromatography on DEAE-cellulose. RESULTS: The mean protein kinase C activity in erythrocytes from healthy individuals was 0.18 +/- 0.02 pmol [32P]phosphate min(-1) x 10(6) cells, regardless of sex and age. The corresponding value for patients with primary hypertension was 0.16 +/- 0.04 pmol [32P]phosphate min(-1) x 10(6) cells. CONCLUSIONS: The amount of protein kinase C, measured as the activity at optimal assay conditions, in erythrocytes from patients with primary hypertension is not critical for the development of moderate hypertension.

Characterization of protein kinase C-mediated phosphorylation of the short cytoplasmic domain isoform of C-CAM.

C-CAM is a ubiquitously expressed cell adhesion molecule belonging to the carcinoembryonic antigen family. Two co-expressed isoforms, C-CAM-L and C-CAM-S, are known, having different cytoplasmic domains both of which can be phosphorylated in vivo. Here we have characterized the PKC-mediated phosphorylation of the short cytoplasmic domain isoform, C-CAM-S. Phorbol myristyl acetate induced phosphorylation of C-CAM-S in transfected CHO cells. Using synthetic peptides and Edman degradation we identified Ser449 as the PKC-phosphorylated amino acid residue. Binding experiments with modified peptides indicated that this phosphorylation decreases the ability of the cytoplasmic domain of C-CAM-S to bind calmodulin.

A potent and highly selective peptide substrate for protein kinase C assay.

Protein kinases exhibit substrate specificities that are often primarily determined by the amino acids around the phosphorylation sites. Peptides corresponding to protein kinase C phosphorylation sites in several different proteins were synthesized on SPOTs membrane which has recently been found to be applicable for studies of protein kinase specificity. After phosphorylation with protein kinase C, we chose the best phosphorylated peptides for the investigation of the importance of amino acids immediately adjacent to the phosphorylation site. The selectivity of the best protein kinase C substrates from this study was analysed with protein kinases A, CK1 and CK2. According to these tests, the most favourable characteristics of SPOTs-membrane-associated peptides were demonstrated by peptide KRAKRKTAKKR. Kinetic analysis of peptide phosphorylation with protein kinase C revealed an apparent Km of 0.49 +/- 0.13 microM and Vmax of 10.0 +/- 0.5 nmol/min per mg with soluble peptide KRAKRKTAKKR. In addition, we assayed several other soluble peptides commonly used as protein kinase C substrates. Peptide KRAKRKTAKKR showed the lowest Km and the highest Vmax/Km value in comparison with peptides FKKSFKL, pEKRPSQRSKYL and KRAKRKTTKKR. Furthermore, of the peptides tested, KRAKRKTAKKR was the most selective substrate for protein kinase C. The favourable kinetic parameters combined with the selectivity should make the KRAKRKTAKKR peptide useful as a substrate for protein kinase C in the assays of both purified enzyme and in crude cell extracts.

Phosphorylation of CaBP1 and CaBP2 by protein kinase CK2.

Several proteins in the mammalian endoplasmic reticulum are substrates for protein kinases. Many unidentified phosphoproteins from this compartment are described in the literature, and this prompted us to try to identify at least the more dominant ones. When solubilized bovine and murine microsomes were phosphorylated with protein kinase CK2 and [32P]ATP and separated on SDS-PAGE, the corresponding autoradiogram showed three dominant 32P-labeled proteins. These three [32P]phosphoproteins were identified as calcium-binding proteins (CaBP) 1, 2, and 4 after purification on a MonoQ column followed by SDS-PAGE, proteolytic cleavage and subsequent amino acid sequencing of the purified 32P-labeled peptides. All three were also phosphorylated by an endogenous kinase, found by us to be of the CK2 type. This kinase phosphorylated CaBP1 N-terminally at serine 427. Of the three proteins, only CaBP4 was previously known to be a substrate of CK2. The newly identified substrates CaBP 1 and 2 are members of the thioredoxin family and have a signal tetrapeptide in the C-terminal of the protein for retention in the ER. Serines and/or threonines in the C-terminal were phosphorylated in CaBP1 when the endogenous CK2 was used as protein kinase. A protein with the same molecular mass as CaBP1 on SDS-PAGE was phosphorylated when intact hepatocytes were grown in the presence of [32P] phosphate. The in vitro phosphorylation with protein kinase CK2 can be used as a specific and sensitive method for identification of CaBP1, 2, and 4 in microsomes.

Phosphorylation of sucrose synthase from maize seedlings.

Two isoforms of sucrose synthase (SS1 and SS2) from maize (Zea mays, var. Mona) seedlings co-purified with a calcium and phospholipid dependent protein kinase. The enzymatic preparation obtained gave a positive reaction with the antibody against mammalian protein kinase C. Maize sucrose synthase was phosphorylated by the endogenous protein kinase. Also, mammalian protein kinases (protein kinase C and protein kinase A) were able to phosphorylate the 86 kDa subunit of sucrose synthase. When excised seedlings were fed [32P]orthophosphate, sucrose synthase was also phosphorylated. Microsequencing of in vivo labelled enzyme has shown phosphorylation of Ser-15 in SS2. The present work provides evidence that maize sucrose synthase is the physiological substrate of the endogenous calcium and phospholipid dependent protein kinase(s).