During long-term cystic fibrosis lung infections, Pseudomonas aeruginosa undergoes genetic adaptation resulting in progressively increased persistence and the generation of adaptive colony morphotypes. This includes small colony variants (SCVs), auto-aggregative, hyper-adherent cells whose appearance correlates with poor lung function and persistence of infection. The SCV morphotype is strongly linked to elevated levels of cyclic-di-GMP, a ubiquitous bacterial second messenger that regulates the transition between motile and sessile, cooperative lifestyles. A genetic screen in PA01 for SCV-related loci identified the yfiBNR operon, encoding a tripartite signaling module that regulates c-di-GMP levels in P. aeruginosa. Subsequent analysis determined that YfiN is a membrane-integral diguanylate cyclase whose activity is tightly controlled by YfiR, a small periplasmic protein, and the OmpA/Pal-like outer-membrane lipoprotein YfiB. Exopolysaccharide synthesis was identified as the principal downstream target for YfiBNR, with increased production of Pel and Psl exopolysaccharides responsible for many characteristic SCV behaviors. An yfi-dependent SCV was isolated from the sputum of a CF patient. Consequently, the effect of the SCV morphology on persistence of infection was analyzed in vitro and in vivo using the YfiN-mediated SCV as a representative strain. The SCV strain exhibited strong, exopolysaccharide-dependent resistance to nematode scavenging and macrophage phagocytosis. Furthermore, the SCV strain effectively persisted over many weeks in mouse infection models, despite exhibiting a marked fitness disadvantage in vitro. Exposure to sub-inhibitory concentrations of antibiotics significantly decreased both the number of suppressors arising, and the relative fitness disadvantage of the SCV mutant in vitro, suggesting that the SCV persistence phenotype may play a more important role during antimicrobial chemotherapy. This study establishes YfiBNR as an important player in P. aeruginosa persistence, and implicates a central role for c-di-GMP, and by extension the SCV phenotype in chronic infections.

The aim of the study was to assess the total energy expenditure (TEE), resting energy expenditure (REE) and physical activity level (PAL) in home-living cachectic patients with advanced pancreatic cancer. The influence of an energy and protein dense oral supplement either enriched with or without the n-3 fatty acid eicosapentaenoic acid (EPA) and administered over an 8-week period was also determined. In total, 24 patients were studied at baseline. The total energy expenditure was measured using doubly labelled water and REE determined by indirect calorimetry. Patients were studied at baseline and then randomised to either oral nutritional supplement. Measurements were repeated at 8 weeks. At baseline, REE was increased compared with predicted values for healthy individuals (1387(42) vs 1268(32) kcal day(-1), P=0.001), but TEE (1732(82) vs 1903(48) kcal day(-1), P=0.023) and PAL (1.24(0.04) vs 1.50) were reduced. After 8 weeks, the REE, TEE and PAL of patients who received the control supplement did not change significantly. In contrast, although REE did not change, TEE and PAL increased significantly in those who received the n-3 (EPA) enriched supplement. In summary, patients with advanced pancreatic cancer were hypermetabolic. However, TEE was reduced and this was secondary to a reduction in physical activity. The control energy and protein dense oral supplement did not influence the physical activity component of TEE. In contrast, administration of the supplement enriched with EPA was associated with an increase in physical activity, which may reflect improved quality of life.

BACKGROUND

The purpose of the study was to compare the effectiveness of olanzapine (OLN) and aprepitant (APR) for the prevention of chemotherapy-induced nausea and vomiting (CINV) in patients receiving highly emetogenic chemotherapy.

METHODS

A phase III trial was performed in chemotherapy-naive patients receiving cisplatin ≥ 70 mg/m(2) or cyclophosphamide ≥ 500 mg/m(2) and doxorubicin ≥ 50 mg/m(2), comparing OLN to APR in combination with palonosetron (PAL) and dexamethasone (DEX). The OLN, PAL, DEX (OPD) regimen was 10 mg of oral OLN, 0.25 mg of IV PAL, and 20 mg of IV DEX prechemotherapy, day 1, and 10 mg/day of oral OLN alone on days 2-4 postchemotherapy. The APR, PAL, DEX (APD) regimen was 125 mg of oral APR, 0.25 mg of IV PAL, and 12 mg of IV DEX, day 1, and 80 mg of oral APR, days 2 and 3, and 4 mg of DEX BID, days 2-4. Two hundred fifty-one patients consented to the protocol and were randomized. Two hundred forty-one patients were evaluable.

RESULTS

Complete response (CR) (no emesis, no rescue) was 97% for the acute period (24 hours postchemotherapy), 77% for the delayed period (days 2-5 postchemotherapy), and 77% for the overall period (0-120 hours) for 121 patients receiving the OPD regimen. CR was 87% for the acute period, 73% for the delayed period, and 73% for the overall period in 120 patients receiving the APD regimen. Patients without nausea (0, scale 0-10, MD Anderson Symptom Inventory) were OPD: 87% acute, 69% delayed, and 69% overall; APD: 87% acute, 38% delayed, and 38% overall. There were no grade 3 or 4 toxicities. CR and control of nausea in subsequent chemotherapy cycles were equal to or greater than cycle 1 for both regimens. OPD was comparable to APD in the control of CINV. Nausea was better controlled with OPD.

CONCLUSIONS

In this study, OLN combined with a single dose of DEX and a single dose of PAL was very effective at controlling acute and delayed CINV in patients receiving highly emetogenic chemotherapy. CR rates were not significantly different from a similar group of patients receiving highly emetogenic chemotherapy and an antiemetic regimen consisting of APR, PAL, and DEX.

The Food and Drug Administration recently approved the use of palivizumab (pal-vizh-mäb), an intramuscularly administered monoclonal antibody preparation. Recommendations for its use are based on a large, randomized study demonstrating a 55% reduction in the risk of hospitalization attributable to respiratory syncytial virus (RSV) infections in high-risk pediatric patients. Infants and children with chronic lung disease (CLD), formerly designated bronchopulmonary dysplasia, as well as prematurely born infants without CLD experienced a reduced number of hospitalizations while receiving palivizumab compared with a placebo. Both palivizumab and respiratory syncytial virus immune globulin intravenous (RSV-IGIV) are available for protecting high-risk children against serious complications from RSV infections. Palivizumab is preferred for most high-risk children because of ease of administration (intramuscular), lack of interference with measles-mumps-rubella vaccine and varicella vaccine, and lack of complications associated with intravenous administration of human immune globulin products. RSV-IGIV, however, provides additional protection against other respiratory viral illnesses and may be preferred for selected high-risk children including those receiving replacement intravenous immune globulin because of underlying immune deficiency or human immuno-deficiency virus infection. For premature infants about to be discharged from hospitals during the RSV season, physicians could consider administering RSV-IGIV for the first month of prophylaxis. Most of the guidelines from the American Academy of Pediatrics for the selection of infants and children to receive RSV-prophylaxis remain unchanged. Palivizumab has been shown to provide benefit for infants who were 32 to 35 weeks of gestation at birth. RSV-IGIV is contraindicated and palivizumab is not recommended for children with cyanotic congenital heart disease. The number of patients with adverse events judged to be related to palivizumab was similar to that of the placebo group (11% vs 10%, respectively); discontinuation of injections for adverse events related to palivizumab was rare.

Phenylalanine ammonia-lyase (PAL) catalyzes the first reaction in the biosynthesis from phenylalanine of a wide variety of phenylpropanoid natural products including lignin, flavonoid pigments, and phytoalexins. In bean (Phaseolus vulgaris L.), PAL is encoded by a family of three genes. We show here by RNase protection with gene-specific probes that these genes are expressed differentially during development and in response to different environmental cues. While all three genes are expressed at high levels in roots, only PALPALPALPALPALPALPALPALPALPALPALPAL polypeptide isoforms were observed by two-dimensional gel electrophoretic analysis of in vitro translation products encoded by RNA isolated from hypocotyls stimulated by light, wounding, or infection. The specific isoforms encoded by transcripts of the three PAL genes were identified by inhibition of synthesis in vitro with gene-specific anti-sense transcripts followed by comparative two-dimensional gel electrophoretic analysis of the pattern of translation products. These data indicate that selective expression of PAL genes encoding functional variants is governed by a complex set of regulatory networks for developmental and environmental control of phenylpropanoid biosynthesis.

To inquire whether the loci identified by recombination-defective and disjunction-defective meiotic mutants in Drosophila are also utilized during mitotic cell division, the effects of 18 meiotic mutants (representing 13 loci) on mitotic chromosome stability have been examined genetically. To do this, meiotic-mutant-bearing flies heterozygous for recessive somatic cell markers were examined for the frequencies and types of spontaneous clones expressing the cell markers. In such flies, marked clones can arise via mitotic recombination, mutation, chromosome breakage, nondisjunction or chromosome loss, and clones from these different origins can be distinguished. In addition, meiotic mutants at nine loci have been examined for their effects on sensitivity to killing by UV and X rays.-Mutants at six of the seven recombination-defective loci examined (mei-9, mei-41, c(3)G, mei-W68, mei-S282, mei-352, mei-218) cause mitotic chromosome instability in both sexes, whereas mutants at one locus (mei-218) do not affect mitotic chromosome stability. Thus many of the loci utilized during meiotic recombination also function in the chromosomal economy of mitotic cells.-The chromosome instability produced by mei-41 alleles is the consequence of chromosome breakage, that of mei-9 alleles is primarily due to chromosome breakage and, to a lesser extent, to an elevated frequency of mitotic recombination, whereas no predominant mechanism responsible for the instability caused by c(3)G alleles is discernible. Since these three loci are defective in their responses to mutagen damage, their effects on chromosome stability in nonmutagenized cells are interpreted as resulting from an inability to repair spontaneous lesions. Both mei-W68 and mei-S282 increase mitotic recombination (and in mei-W68, to a lesser extent, chromosome loss) in the abdomen but not the wing. In the abdomen, the primary effect on chromosome stability occurs during the larval period when the abdominal histoblasts are in a nondividing (G2) state.-Mitotic recombination is at or above control levels in the presence of each of the recombination-defective meiotic mutants examined, suggesting that meiotic and mitotic recombination are under separate genetic control in Drosophila.-Of the six mutants examined that are defective in processes required for regular meiotic chromosome segregation, four (l(1)TW-6(cs), ca(nd), mei-S332, ord) affect mitotic chromosome behavior. At semi-restrictive temperatures, the cold sensitive lethal l(1)TW-6(cs) causes very frequent somatic spots, a substantial proportion of which are attributable to nondisjunction or loss. Thus, this locus specifies a function essential for chromosome segregation at mitosis as well as at the first meiotic division in females. The patterns of mitotic effects caused by ca(nd), mei-S332, and ord suggest that they may be leaky alleles at essential loci that specify functions common to meiosis and mitosis. Mutants at the two remaining loci (nod, pal) do not affect mitotic chromosome stability.

Phylogenetic Analysis Library (PAL) is a collection of Java classes for use in molecular evolution and phylogenetics. PAL provides a modular environment for the rapid construction of both special-purpose and general analysis programs. PAL version 1.1 consists of 145 public classes or interfaces in 13 packages, including classes for models of character evolution, maximum-likelihood estimation, and the coalescent, with a total of more than 27000 lines of code. The PAL project is set up as a collaborative project to facilitate contributions from other researchers. AVAILIABILTY: The program is free and is available at http://www.pal-project.org. It requires Java 1.1 or later. PAL is licensed under the GNU General Public License.

Phenylalanine ammonia-lyase (PAL) catalyses the first step in the biosynthesis of phenylpropanoids, which form a wide variety of plant secondary products. The transcription of PAL is regulated in response to various factors that induce the accumulation of flavonoids, lignin and compounds thought to be involved in plant defence reactions. The 5' upstream sequence of a PAL gene from Phaseolus vulgaris was fused to the coding region of the reporter gene encoding beta-glucuronidase (GUS), and transformed into potato and tobacco plants. Histochemical analysis of GUS expression showed that the PAL promoter was active in specific cell types that accumulated phenylpropanoid derivatives in response to mechanical wounding, and also during normal development of the xylem and flower. In xylem that had undergone secondary thickening, GUS activity occurred in rays of cells thought to be the xylem parenchyma. It was postulated that PAL activity in these cells could provide intermediates for lignin synthesis in xylem vessels that had terminally differentiated.

Alveolar macrophages (AMs) avidly bind and ingest unopsonized environmental particles and bacteria through scavenger-type receptors (SRs). AMs from mice with a genetic deletion of the major macrophage SR (types AI and AII; SR-/-) showed no decrease in particle binding compared with SR+/+ mice, suggesting that other SRs are involved. To identify these receptors, we generated a monoclonal antibody (mAb), PAL-1, that inhibits hamster AM binding of unopsonized particles (TiO2, Fe2O3, and latex beads; 66 +/- 5, 77 +/- 2, and 85 +/- 2% inhibition, respectively, measured by flow cytometry). This antibody identifies a protein of approximately 70 kD on the AM surface (immunoprecipitation) that is expressed by AMs and other macrophages in situ. A cDNA clone encoding the mAb PAL-1-reactive protein isolated by means of COS cell expression was found to be 84 and 77% homologous to mouse and human scavenger receptor MARCO mRNA, respectively. Transfection of COS cells with MARCO cDNA conferred mAb-inhibitable TiO2 binding. Hamster MARCO also mediates AM binding of unopsonized bacteria (67 +/- 5 and 47 +/- 4% inhibition of Escherichia coli and Staphylococcus aureus binding by mAb PAL-1). A polyclonal antibody to human MARCO identified the expected approximately 70-kD band on Western blots of lysates of normal bronchoalveolar lavage (BAL) cells (>90% AMs) and showed strong immunolabeling of human AMs in BAL cytocentrifuge preparations and within lung tissue specimens. In normal mouse AMs, the anti-MARCO mAb ED31 also showed immunoreactivity and inhibited binding of unopsonized particles (e.g., TiO2 approximately 40%) and bacteria. The novel function of binding unopsonized environmental dusts and pathogens suggests an important role for MARCO in the lungs' response to inhaled particles.

It has been reported that among drugs with mixed actions on central nervous system monoamine systems, increased serotonergic activity is associated with decreased potency as a reinforcer. The present experiment was designed to examine this relationship for amphetamine analogs that varied in serotonin releasing potency and to evaluate whether serotonergic actions can affect reinforcing efficacy. Compounds PAL 313 and 314 are para- and meta-methylamphetamine, respectively. PAL 303 and 353 are para- and meta-fluoroamphetamine, respectively. All compounds had similar potencies as in vitro releasers of dopamine (DA) and norepinephrine (NE) but differed in potency for 5-hydroxytryptamine (serotonin) (5-HT) release [EC(50) (nanomolar) PAL 313 = 53.4; PAL 314 = 218; PAL 303 = 939; PAL 353 = 1937]. When made available to rhesus monkeys (Macaca mulatta)(n = 4) for self-administration under a fixed-ratio 25 schedule, all were positive reinforcers with biphasic dose-response functions (0.003-1.0 mg/kg) and were equipotent. PAL 313 was self-administered at a lower rate than the other compounds, which were indistinguishable. Under a progressive-ratio schedule (n = 5), all drugs were positive reinforcers. Dose-response functions increased to a maximum or were biphasic (0.01-1.0 mg/kg), and drugs were equipotent. At maximum, PAL 313 maintained less responding than other PAL drugs, which maintained similar maxima. Thus, all compounds were positive reinforcers under both schedules, consistent with their potent DA actions. Responding was lower when 5-HT potency was higher and comparable with DA and NE potency. The results suggest that the mechanism for this effect involves a decrease in reinforcing potency and efficacy among monoamine releasing agents when 5-HT releasing potency is increased relative to DA.

The apposed membranes of myelinating Schwann cells are joined by several types of junctional specializations known as autotypic or reflexive junctions. These include tight, gap, and adherens junctions, all of which are found in regions of noncompact myelin: the paranodal loops, incisures of Schmidt-Lanterman, and mesaxons. The molecular components of autotypic tight junctions have not been established. Here we report that two homologues of Discs Lost-multi PDZ domain protein (MUPP)1, and Pals-associated tight junction protein (PATJ), are differentially localized in myelinating Schwann cells and associated with different claudins. PATJ is mainly found at the paranodal loops, where it colocalized with claudin-1. MUPP1 and claudin-5 colocalized in the incisures, and the COOH-terminal region of claudin-5 interacts with MUPP1 in a PSD-95/Disc Large/zona occludens (ZO)-1 (PDZ)-dependent manner. In developing nerves, claudin-5 and MUPP1 appear together in incisures during the first postnatal week, suggesting that they coassemble during myelination. Finally, we show that the incisures also contain four other PDZ proteins that are found in epithelial tight junctions, including three membrane-associated guanylate-kinase proteins (membrane-associated guanylate-kinase inverted-2, ZO-1, and ZO-2) and the adaptor protein Par-3. The presence of these different tight junction proteins in regions of noncompact myelin may be required to maintain the intricate cytoarchitecture of myelinating Schwann cells.

Light-activated ion channels provide a precise and noninvasive optical means for controlling action potential firing, but the genes encoding these channels must first be delivered and expressed in target cells. Here we describe a method for bestowing light sensitivity onto endogenous ion channels that does not rely on exogenous gene expression. The method uses a synthetic photoisomerizable small molecule, or photoswitchable affinity label (PAL), that specifically targets K+ channels. PALs contain a reactive electrophile, enabling covalent attachment of the photoswitch to naturally occurring nucleophiles in K+ channels. Ion flow through PAL-modified channels is turned on or off by photoisomerizing PAL with different wavelengths of light. We showed that PAL treatment confers light sensitivity onto endogenous K+ channels in isolated rat neurons and in intact neural structures from rat and leech, allowing rapid optical regulation of excitability without genetic modification.

Extremes of pH are an occupational hazard for many microorganisms. In addition to efficient pH homeostasis, survival effectively requires a regulatory system tailoring the syntheses of molecules functioning beyond the cell boundaries (permeases, secreted enzymes, and exported metabolites) to the pH of the growth environment. Our previous work established that the zinc finger PacC transcription factor mediates such pH regulation in the fungus Aspergillus nidulans in response to a signal provided by the products of the six pal genes at alkaline ambient pH. In the presence of this signal, PacC becomes functional, activating transcription of genes expressed at alkaline pH and preventing transcription of genes expressed at acidic pH. Here we detect two forms of PacC in extracts, both forming specific retardation complexes with a PacC-binding site. Under acidic growth conditions or in acidity-mimicking pal mutants (defective in ambient pH signal transduction), the full-length form of PacC predominates. Under alkaline growth conditions or in alkalinity-mimicking pacCc mutants (independent of the ambient pH signal), a proteolysed version containing the amino-terminal approximately 40% of the protein predominates. This specifically cleaved shorter version is clearly functional, both as an activator for alkaline-expressed genes and as a repressor for acid-expressed genes, but the full-length form of PacC must be inactive. Thus, PacC proteolysis is an essential and pH-sensitive step in the regulation of gene expression by ambient pH. Carboxy-terminal truncations, resulting in a gain-of-function (pacCc) phenotype, bypass the requirement for the pal signal transduction pathway for conversion of the full-length to the proteolyzed functional form.

The tremendous dynamics of HIV infection finds expression in the tempo of sequence diversification. Genetic diversity calculations require the clearance of a majority of infected cells, the obvious predator being anti-HIV immune responses. Indeed, infiltration of germinal centers (GCs) by HIV-specific CD8(+) cytotoxic T lymphocytes has been described. A corollary to this description would be limited diffusion of virus within lymphoid structures. HIV efficiently infects and replicates mainly in activated CD4(+) T lymphoblasts. These cells are found within GCs after their activation in the adjacent periarteriolar lymphoid sheath (PALS). Here GCs and PALS have been dissected from consecutive 10-micrometer sections through splenic tissue from three HIV-1-infected patients. Nested PCR amplification of the two first hypervariable regions of the env gene indicated that 38-78% of sections contained HIV-infected cells. Since there are several hundred CD4(+) T cells per GC section, approximately 0.09-0.64% harbor proviral DNA. Such a low frequency not only suggests that virions on the follicular dendritic cell surfaces do not readily infect adjacent T cells but also indicates highly restricted spread of HIV within GCs and the PALS. Sections were heavily infiltrated by CD8(+) cells, which, together with a large body of extant data, suggests that the majority of infected cells are destroyed by HIV-specific cytotoxic T lymphocytes before becoming productively infected. Finally, sequence analysis revealed that those HIV-positive cells were multiply infected, which helps explain widespread recombination despite a low overall frequency of infected cells.

OBJECTIVE

Obesity results from protracted energy imbalance. Whether this comprises excessive energy intake, lowered physical activity or both, remains disputed.

METHODS

Physical activity energy expenditure, evaluated in three different ways from daily energy expenditure (DEE) measured using doubly labelled water, was examined for trends over time. Data included subjects in Europe (Maastricht, the Netherlands) and North America extending back to the 1980s. These data were compared with measures from the third world, and measures made on wild terrestrial mammals.

RESULTS

Physical activity expenditure in Europe (residual of the regression of DEE on basal energy expenditure (BEE)) has slightly but significantly increased since the 1980s. There was no trend over time in physical activity level (PAL=DEE/BEE), or in the residual variance in DEE once mass, sex and age were accounted for. This latter index of physical activity expenditure also significantly increased over time in North America. DEE of individuals in Europe and North America was not significantly different from individuals measured in the third world. In wild terrestrial mammals, DEE mostly depended on body mass and ambient temperature. Predicted DEE for a 78 kg mammal living at 20 degrees C was 9.2 MJ per day (95% CI: 7.9-12.9 MJ per day), not significantly different from the measured DEE of modern humans (around 10.2-12.6 MJ per day).

CONCLUSIONS

As physical activity expenditure has not declined over the same period that obesity rates have increased dramatically, and daily energy expenditure of modern man is in line with energy expenditure in wild mammals, it is unlikely that decreased expenditure has fuelled the obesity epidemic.

This study investigated the relationship between delayed-onset muscle soreness and other indicators of muscle damage following eccentric exercise. Male students (n = 110) performed 12 (12ECC), 24 (24ECC), or 60 maximal eccentric actions of the elbow flexors (60ECC). Maximal isometric force, relaxed and flexed elbow joint angles, upper arm circumference, and plasma creatine kinase activity were assessed immediately before and after, and for 4 days after exercise. Muscle soreness (SOR) was evaluated by a visual analog scale (a 50-mm line, 0: no pain, 50: extremely painful) when the elbow flexors were palpated (SOR-Pal), flexed (SOR-Flx) and stretched (SOR-Ext). Although 24ECC and 60ECC resulted in significantly (P <; 0.05) larger changes in all indicators and slower recovery compared to 12ECC, no significant differences were evident for SOR-Pal and SOR-Flx between 12ECC and 24ECC, or 12ECC and 60ECC. In contrast, SOR-Ext was significantly (P <; 0.05) lower for 12ECC compared to 24ECC and 60ECC. A Pearson product-moment correlation showed SOR-Pal did not correlate significantly with any indicators, however, SOR-Ext and SOR-Flx showed weak (r <; 0.32) but significant (P <; 0.05) correlations with other indicators. Because of generally poor correlations between DOMS and other indicators, we conclude that use of DOMS is a poor reflector of eccentric exercise-induced muscle damage and inflammation, and changes in indirect markers of muscle damage and inflammation are not necessarily accompanied with DOMS.

The spleen of rats and mice was studied with the light and electron microscope. Special attention was paid to the delineation and composition of the white pulp compartments: periarteriolar lymphatic sheath (PALS), follicles and marginal zone. These three compartments each have their specific lymphoid and non-lymphoid cells. Reticulum cells and reticulin fibres, although occurring in all three compartments, from a characteristic pattern in each compartment. In the PALS two areas can be distinguished: a central area, largely devoid of reticulum cells, and a peripheral area where reticulum cells are arranged in cylindrical shells. The central PALS FORMS THE THYMUS DEPENDENT AREA OF THE SPLEEN, THE PERIPHERAL PALS contains both T- and B-lymphocytes. T-B-interactions requiring cell contact could take place in the latter area. Lymph vessels originate from the shells of reticulum cells around the smaller arterioles; these vessels follow the central arteriole to the hilus of the spleen. Circumstantial evidence suggests that the lymph vessels form a recirculation pathway for T-cells and possibly also for B-cells. In two areas of the splenic white pulp characteristic non-lymphoid cells are present. The central PALS contains interdigitating cells (IDC), which show a close contact with surrounding T-lymphocytes. The light zone of the follicle centre exhibits dendritic cells (DC). B-cells are found between the ramifications of the DC. It is conceivable that these cells play a role in the homing of T-cells and B-cells respectively. In addition they might create a microenvironment supporting differentiation and proliferation of T- and B-cells. The marginal zone does not contain a characteristic non-lymphoid cell type. However, in this compartment B-cells are directly exposed to the circulating blood. It is suggested that this factor constitutes one of the essentials of the microenvironment in the marginal zone.

The antigenic status of vascular endothelium from different sites of the normal adult and fetal human cardiovascular system was investigated. Tissues included aorta (n = 9), pulmonary artery (n = 8), coronary artery (n = 6), ventricle/atrium (n = greater than 10), lymph node (n = 2), fetal whole heart (n = 3), and umbilical cord (n = 7). Frozen sections were studied using monoclonal antibodies recognizing endothelial markers (EN4, vWf, Pal-E, and 44G4), vascular adhesion molecules (ICAM-1, ELAM, VCAM, and PECAM), the monocyte/endothelial marker (OKM5), and major histocompatibility complex (MHC) molecules (class I and class II). Results demonstrate that capillary endothelium is phenotypically different from endothelial cells (EC) lining large vessels. Capillary EC strongly express MHC classes I and II, ICAM, and OKM5, which are variably weak to undetectable on large vessels. In contrast, the large vessels strongly express vWf and appear to constitutively express ELAM-1. This suggests that the capillary EC may be more efficient at antigen presentation or more susceptible to immune attack in vivo. Interestingly, normal coronary arteries, unlike all other large vessels, express MHC class II and VCAM molecules. Future studies should concentrate on comparative functional studies between capillary, coronary, and large vessel EC.

We have isolated a cDNA for a cytochrome P450, cinnamate 4-hydroxylase (C4H), of Arabidopsis thaliana using a C4H cDNA from mung been as a hybridization probe. The deduced amino acid sequence is 84.7% identical to that of mung bean C4H and therefore was designated CYP73A5. The CYP73A5 protein was expressed in insect cells using the baculovirus expression system and when reconstituted with lipid and NADPH-cytochrome P450 reductase resulted in C4H activity with a specific activity of 68 nmol min-1 nmol-1 P450. Southern blot analysis revealed that CYP73A5 is a single-copy gene in Arabidopsis. C4H (CYP73A5) expression was apparently coordinated in Arabidopsis with both PALPALPALPAL and 4CL genes so far reported as controlling expression.

Investigations using wearable monitors have begun to examine how sedentary time behaviors influence health.

OBJECTIVE

The objective of this study is to demonstrate the use of a measure of sedentary behavior and to validate the activPAL (PAL Technologies Ltd., Glasgow, Scotland) and ActiGraph GT3X (Actigraph, Pensacola, FL) for estimating measures of sedentary behavior: absolute number of breaks and break rate.

METHODS

Thirteen participants completed two 10-h conditions. During the baseline condition, participants performed normal daily activity, and during the treatment condition, participants were asked to reduce and break up their sedentary time. In each condition, participants wore two ActiGraph GT3X monitors and one activPAL. The ActiGraph was tested using the low-frequency extension filter (AG-LFE) and the normal filter (AG-Norm). For both ActiGraph monitors, two count cut points to estimate sedentary time were examined: 100 and 150 counts per minute. Direct observation served as the criterion measure of total sedentary time, absolute number of breaks from sedentary time, and break rate (number of breaks per sedentary hour (brk·sed-h)).

RESULTS

Break rate was the only metric sensitive to changes in behavior between baseline (5.1 [3.3-6.8] brk·sed-h) and treatment conditions (7.3 [4.7-9.8] brk·sed-h) (mean (95% confidence interval)). The activPAL produced valid estimates of all sedentary behavior measures and was sensitive to changes in break rate between conditions (baseline, 5.1 [2.8-7.1] brk·sed-h; treatment, 8.0 [5.8-10.2] brk·sed-h). In general, the AG-LFE and AG-Norm were not accurate in estimating break rate or the absolute number of breaks and were not sensitive to changes between conditions.

CONCLUSIONS

This study demonstrates the use of expressing breaks from sedentary time as a rate per sedentary hour, a metric specifically relevant to free-living behavior, and provides further evidence that the activPAL is a valid tool to measure components of sedentary behavior in free-living environments.

Genes encoding phenylalanine ammonia-lyase (<em>PAL</em>) are expressed in a complex pattern during plant development and in response to light, pathogen ingress, mechanical damage and other stresses. Analysis of the promoter of the bean <em>PAL</em>2 gene in transgenic tobacco has shown that some regions responsible for developmental expression are functionally compensatory. The minimum sequence containing all cis sequences necessary for developmental patterns of expression is within -254 bp of the transcription start site. Footprinting and electrophoretic mobility shift assay studies of this region revealed potential cis sequences which coincided with the functional domains defined by small deletions and promoter fusions. Mutations in these potential cis sequences in the context of the minimal -254 bp promoter altered tissue-specific expression patterns, confirming the importance of these sequences for expression in vivo. A functional model for the promoter is presented which predicts that three AC-elements, which are possible Myb protein binding sites, together with a G-box, interact to direct the complex patterns of tissue-specific expression observed.

The miniaturization of biological and chemical analytical devices by micro-electro-mechanical-systems (MEMS) technology has posed a vital influence on such fields as medical diagnostics, microbial detection and other bio-analysis. Among many miniaturized analytical devices, the polymerase chain reaction (PCR) microchip/microdevices are studied extensively, and thus great progress has been made on aspects of on-chip micromachining (fabrication, bonding and sealing), choice of substrate materials, surface chemistry and architecture of reaction vessel, handling of necessary sample fluid, controlling of three or two-step temperature thermocycling, detection of amplified nucleic acid products, integration with other analytical functional units such as sample preparation, capillary electrophoresis (CE), DNA microarray hybridization, etc. However, little has been done on the review of above-mentioned facets of the PCR microchips/microdevices including the two formats of flow-through and stationary chamber in spite of several earlier reviews [Zorbas, H. Miniature continuous-flow polymerase chain reaction: a breakthrough? Angew Chem Int Ed 1999; 38 (8):1055-1058; Krishnan, M., Namasivayam, V., Lin, R., Pal, R., Burns, M.A. Microfabricated reaction and separation systems. Curr Opin Biotechnol 2001; 12:92-98; Schneegabeta, I., Köhler, J.M. Flow-through polymerase chain reactions in chip themocyclers. Rev Mol Biotechnol 2001; 82:101-121; deMello, A.J. DNA amplification: does 'small' really mean 'efficient'? Lab Chip 2001; 1: 24N-29N; Mariella, Jr. R. MEMS for bio-assays. Biomed Microdevices 2002; 4 (2):77-87; deMello AJ. Microfluidics: DNA amplification moves on. Nature 2003; 422:28-29; Kricka, L.J., Wilding, P. Microchip PCR. Anal BioAnal Chem 2003; 377:820-825]. In this review, we survey the advances of the above aspects among the PCR microfluidic devices in detail. Finally, we also illuminate the potential and practical applications of PCR microfluidics to some fields such as microbial detection and disease diagnosis, based on the DNA/RNA templates used in PCR microfluidics. It is noted, especially, that this review is to help a novice in the field of on-chip PCR amplification to more easily find the original papers, because this review covers almost all of the papers related to on-chip PCR microfluidics.

Phenylalanine ammonia-lyase (<em>PAL</em>; EC 4.3.1.5) genomic sequences were isolated from bean (Phaseolus vulgaris L.) genomic libraries using elicitor-induced bean <em>PAL</em> cDNA sequences as a probe. Southern blot hybridization of genomic DNA fragments revealed three divergent classes of <em>PAL</em> genes in the bean genome. Polymorphic forms were observed within each class. The nucleotide sequences of two <em>PAL</em> genes, g<em>PAL</em>2 (class II) and g<em>PAL</em>3 (class III), were determined. g<em>PAL</em>2 contains an open reading frame encoding a polypeptide of 712 amino acids, interrupted by a 1720 bp intron in the codon for amino acid 130. g<em>PAL</em>3 encodes a polypeptide of 710 amino acids showing 72% similarity with that encoded by g<em>PAL</em>2, and contains a 447 bp intron at the same location. At the nucleotide level, g<em>PAL</em>2 and g<em>PAL</em>3 show 59% sequence similarity in exon I, 74% similarity in exon II, and extensive sequence divergence in the intron, 5' and 3' flanking regions. S1 nuclease protection identified transcription start sites of g<em>PAL</em>2 and g<em>PAL</em>3 respectively 99 bp and 35 bp upstream from the initiation codon ATG, and showed that g<em>PAL</em>2 but not g<em>PAL</em>3 was activated by elicitor, whereas both were activated by wounding of hypocotyls. The 5' flanking region of both genes contain TATA and CAAT boxes, and sequences resembling the SV40 enhancer core. g<em>PAL</em>2 contains a 40 bp palindromic sequence and a 22 bp motif that are also found at similar positions relative to the TATA box in 5' flanking regions of other elicitor-induced bean genes.

The outer membrane of gram-negative bacteria acts as a barrier against harmful lipophilic compounds and larger molecules unable to diffuse freely through the porins. However, outer membrane proteins together with the Tol-Pal and TonB systems have been exploited for the entry of macromolecules such as bacteriocins and phage DNA through the Escherichia coli cell envelope. The TonB system is involved in the active transport of iron siderophores and vitamin B12, while no more precise physiological role of the Tol-Pal system has yet been defined than its requirement for cell envelope integrity. These two systems, containing an energized inner membrane protein interacting with outer membrane proteins, share similarities.