OBJECTIVE

It has been suggested that regular physical activity might maintain and promote the antioxidant defense capacity against oxidative stress. Therefore, we assessed exercise-induced oxidative stress in relation to habitual physical activity level (PAL) in older adults.

METHODS

The study included a 2-week observation period for the measurement of average daily metabolic rate (ADMR) and PAL. Exercise-induced oxidative stress was measured during a 45-minute cycling test at submaximal intensity.

METHODS

A university medical research center.

METHODS

Twenty-six subjects volunteered for the study (n = 26; mean age +/- standard deviation 60 +/- 1; body mass index 27 +/- 1 kg/m2).

METHODS

PAL was determined as ADMR combined with a measurement of basal metabolic rate (BMR): PAL = ADMR/BMR. ADMR was measured over 2 weeks with the doubly labeled water method, preceded by a BMR measurement with a ventilated hood. Antipyrine oxidation was used as marker for oxidative stress in vivo. Reaction of antipyrine with hydroxyl radicals results in the formation of para-hydroxyantipyrine (p-APOH) and ortho-hydroxyantipyrine (o-APOH), where o-APOH is not formed through alternative oxygenetic pathways.

RESULTS

PAL was inversely related to the exercise-induced increase in the ratio of o-APOH to native antipyrine (r = 0.49, P = .010). The relationship between PAL and exercise-induced increase in the ratio of p-APOH (r = 0.30, P = .140) or thiobarbituric acid reactive species (r = 0.31, P = .130) did not reach the level of significance.

CONCLUSIONS

Physically active older adults have a reduced exercise-induced oxidative stress than older adults with a lower level of physical activity. It seems that regular physical activity improves the antioxidant defense capacity.

In an attempt to reveal the role of antigen-laden marginal metallophil (MM) and other macrophages in the intrasplenic immune response of a specific B-cell lineage to a thymus-independent type-2 antigen (Ficoll conjugated with fluorescein isothiocyanate), simultaneous immunohistological observations of the involved cells were performed in the rat. By newly established methods of double or triple immunostainings, time-kinetics of the following parameters were studied and compared: (1) the antigen, (2) the specific antibody-forming cells (AFC) directed to the fluorescein-isothiocyanate determinant, (3) proliferating cells labeled with 5-bromo-2'-deoxyuridine (BrdU), and (4) macrophage subpopulations recognized by monoclonal antibodies (ED2 and ED3). The antigen localized stably not only in the marginal-zone macrophages but also in the MM except around the follicular area. The increase of BrdU-positive cells was observed from day 2 up to day 4 after antigen injection mostly in the periphery of the periarterial lymphoid sheath (outer PALS), which indicated antigen-induced proliferation. As a novel finding, the majority of AFC, both BrdU-positive and -negative, were either closely associated with the antigen-laden MM, or forming cell clusters with ED2-positive macrophages in the outer PALS. In contrast, there were very few AFC in juxtaposition to antigen-free MM in the follicular area or the antigen-laden marginal zone macrophages. The results led to the proposal of a hypothesis that the antigen-laden MM together with ED2-positive macrophages constitute an immunoproliferative microenvironment for the plasmacellular reaction by accumulating the antigen-specific B-cell lineage and promoting these cells to differentiate into the AFC and to proliferate in the outer PALS.

We analyzed the developmental regulation and the activation by wounding of several stress-related genes in various parsley organs. The genes encode phenylalanine ammonia-lyase (PAL) and 4-coumarate:CoA ligase (4CL), two enzymes of general phenylpropanoid metabolism; a flavonoid specific enzyme, chalcone synthase (CHS); a furanocoumarin specific enzyme, bergaptol O-methyltransferase (BMT); and a pathogenesis-related protein (PR 1). All genes or gene families exhibited high levels of expression in roots and during certain stages of leaf development. PAL, 4CL and CHS were preferentially expressed in young leaves, BMT and PR 1 in old leaves. An appreciable increase in CHS mRNA levels was observed in wounded leaves. By contrast, root wounding led to a decrease in the existing CHS mRNA levels. A biphasic response (a decrease followed by an increase) to wounding was seen for BMT and PR 1 mRNAs in roots and for BMT mRNA in attached leaves. Using gene-specific oligonucleotide probes to measure the expression rates of three of the four PAL genes and of the two 4CL genes separately we observed a differential behavior of the individual family members under many of the conditions tested. While PAL-3 was preferentially activated in wounded leaves and 4CL-1 in wounded roots, PAL-2 and 4CL-2 were primarily responsible for the high constitutive expression levels in roots and flowering stems respectively. Despite the differential expression of their individual members, the PAL and 4CL gene families displayed very similar changes in the overall patterns of expression, reflecting their closely related functions in phenylpropanoid metabolism.

The heterogeneity of tissue-specific manifestations of generalized resistance to thyroid hormone (GRTH) could result from differential interactions between the mutant thyroid hormone (T3) receptor-beta (TR beta) on T3 response elements (TREs) in different T3-responsive genes. To explore this hypothesis, the mutant TR beta associated with kindred A, P448H; a TR beta mutant, P448L; and a comparable TR alpha mutant (P398H) were tested for intrinsic function and for inhibition of wild-type TR alpha- and -beta-induced expression from four structurally distinct TREs, the rGH ABC*, the rGH palindrome (PAL), the rat malic enzyme (ME), and the chicken lysozyme silencer F2 (F2). The relative function of the mutants was similarly reduced on the four TREs studied and was T3 concentration dependent. The TR alpha mutant retained the intrinsically greater potency characteristic of this isoform, but remained impaired with respect to wild-type TR alpha even at 500 nM T3. In general, dominant negative inhibition of wild-type TR alpha and -beta function was dependent upon the T3 concentration, as expected from the decreased affinity for ligand conferred by this mutation. A T3 concentration sufficient to relieve the inhibition of wild-type TR function on the ABC*, PAL, and ME TREs (50 nM) had no effect on inhibition of the F2 TRE by the mutant TRs. Receptor isoform preferential inhibition was observed on the ABC*, PAL, and ME TREs by the mutant TRs. Thus, both TRE structure and the isoform of endogenously active receptor could determine the degree of inhibition of a specific gene in GRTH individuals. Further, the lack of dominant negative potentials does not explain the absence of TR alpha mutations in GRTH kindreds.

Munc13 proteins are essential regulators of synaptic vesicle priming and play a key role in adaptive synaptic plasticity phenomena. We recently identified and characterized the Ca(2+)-dependent interaction of Munc13 and calmodulin (CaM) as the molecular mechanism linking changes in residual Ca(2+) concentrations to presynaptic vesicle priming and short-term plasticity. Here, we used peptidic photoprobes covering the established CaM-binding motif of Munc13 for photoaffinity labeling (PAL) of CaM, followed by structural characterization of the covalent photoadducts. Our innovative analytical workflow based on isotopically labeled CaM and mass spectrometry revealed that, in the bound state, the hydrophobic anchor residue of the CaM-binding motif in Munc13s contacts two distinct methionine residues in the C-terminal domain of CaM. To address the orientation of the peptide during binding, we obtained additional distance constraints from the mass spectrometric analysis of chemically cross-linked CaM-Munc13 peptide adducts. The constraints from both complementary cross-linking approaches were integrated into low-resolution three-dimensional structure models of the CaM-Munc13 peptide complexes. Our experimental data are best compatible with the structure of the complex formed by CaM and a CaM-binding peptide derived from neuronal NO synthase and show that Munc13-1 and ubMunc13-2 bind to CaM in an antiparallel orientation through a 1-5-8 motif. The structural information about the CaM-Munc13 peptide complexes will facilitate the design of Munc13 variants with altered CaM affinity and thereby advance the detailed functional analysis of the role of Munc13 proteins in synaptic transmission and plasticity.

Colicins and phages parasitize outer membrane receptors whose physiological purpose is the transport of metabolites, metals, vitamins, and sugars. From mutagenesis studies, it is known that several colicins require the function of two outer membrane protein (Omp) receptors for cytotoxicity. A formidable list of problems associated with an understanding of a two receptor mechanism for colicin translocation includes the definition of the sites of initial binding and interactions of the colicin with the OM translocator protein, the working lumenal aperture of the translocator, the question of whether the colicin must be unfolded for translocation, the source of energy for unfolding and translocation, the order of colicin translocation, and the sites and mechanism of interaction of the colicins with the Tol-Pal proteins on the periplasmic side of the outer membrane. 3D crystal structures recently obtained of the cobalamin (vitamin B12) receptor (BtuB), and of the complex of BtuB with the 135 residue receptor binding domain (R135) of colicin E3, have provided some new insights on the interactions between two Omp receptors that are necessary for translocation of colicins. Together with spectroscopic data on the R135-BtuB interaction and electrophysiological data on the colicin E3-OmpF interaction, this has led to a proposal for the utilization of two receptors, BtuB-OmpF, in an outer membrane translocon for colicin E3.

Complementary DNAs for two mutant thyroid hormone alpha1 receptors (TR alpha1) were isolated from hepatocellular carcinomas of two patients. Sequence analyses of the complementary DNAs showed a single Val390Ala and double Pro398Ser/Glu350Lys mutations in mutants H and L, respectively. We characterized their hormone-binding, DNA-binding, and dominant negative activities. Mutants H and L did not bind the hormone T3. Their DNA-binding activities were analyzed using three types of thyroid hormone response elements (TREs) in which the half-site binding motifs are arranged in an everted repeat (Lys), an inverted repeat (Pal), or a direct repeat separated by four nucleotides (DR4). Compared with wild-type TR alpha1 (w-TR alpha1), which bound these TREs with different homodimer/monomer ratios, binding of mutant L to the three TREs as homodimers was reduced by approximately 90%. However, binding of mutant H to these TREs was more complex. Although it bound normally to DR4 as homodimers, its binding to Lys as homodimers was reduced by approximately 80%. Surprisingly, its binding to Pal was markedly enhanced compared with w-TR alpha1. The binding of these two mutants to the three TREs as heterodimers with retinoid X receptors (RXR alpha and -beta) was not significantly affected. Consistent with the lack of T3-binding activity, both mutants had lost their trans-activation capacity. Mutants H and L exhibited dominant negative activity, but differed in their TRE dependency. The dominant negative potency of mutant H was in the rank order of Pal>> DR4>> Lys, whereas no TRE dependency was observed for mutant L. The present study indicates that mutations of the TR alpha gene do occur in patients and that these novel TR alpha1 mutants provide a valuable tool to further understand the molecular basis of the dominant negative action of mutant TRs.

Human genetics researchers have been intrigued for many years by weak-to-moderate associations between markers and diseases. However, in most cases of association, the cause of this phenomenon is still not known. Recently, interest has grown in pursuing association studies for complex diseases, either instead of or in addition to linkage studies. Hence, it is timely to reconsider what a disease-marker association, particularly in the weak-to-moderate range (relative risk < 10), can tell us about disease etiology. To this end, this study accomplishes three aims: (1) It formulates two different models explaining weak-to-moderate associations and derives the relationship between them. One is a linkage disequilibrium model, and the other is a "susceptibility," or pure association, model. The importance of drawing the distinction between these two models and the implications for our understanding of the genetics of human disease will also be discussed. It will be argued that the linkage disequilibrium model represents true linkage but that the susceptibility model does not. (2) It examines two family-based association tests proposed recently by Parsian et al. and Spielman et al. and derives formulas for their behavior under the two models described above. It demonstrates that these tests yield almost identical results under these two models. It shows that, whereas these tests can confirm an association, they cannot determine whether the association is caused by the linkage disequilibrium model or the susceptibility model. The study also characterizes the probabilities yielded by the family association tests in the presence of weak-to-moderate associations, which will aid researchers using these tests. (3) It proposes two approaches, both based on linkage analysis, which can distinguish between the two models described above. One approach involves a straightforward linkage analysis of the data; the other involves a partitioned association-linkage (PAL) test, as suggested by Greenberg. Formulas are derived for testing identity by descent in affected sib pairs by using both approaches. (4) Finally, the formulas and arguments are illustrated with two examples from the literature and one computer-simulated data set.

Many human gene therapies will require cell-specific targeting. Though recombinant viruses are much more efficient than nonviral vectors, the latter, especially polymers, have the advantage of being targetable via conjugation of cell-specific ligands, including sugars, peptides, and antibodies, which can be covalently attached to the polymer using a variety of chemistries. Cyclodextrin, which forms inclusion complexes with small hydrophobic molecules, has been incorporated into a gene-delivery polymer and may provide a facile and versatile attachment site for targeting ligands. Polyethylenimine (PEI) was derivatized with beta-cyclodextrin on approximately 10% of the polymer's amines (termed CD-PEI). Human insulin was also derivatized with a hydrophobic palmitate group (pal-HI), which could anchor the protein to CD-PEI/DNA polyplexes. CD-PEI was essentially nontoxic to HEK293 cells at concentrations optimal for gene delivery and mediated nearly 4-fold higher gene expression than unmodified PEI, which is relatively toxic to these cells. More importantly, addition of the pal-HI to CD-PEI enhanced gene expression by more than an order of magnitude compared to unmodified PEI, either with or without the pal-HI. Because of the relative ease with which CD-binding moieties may be attached to various types of ligands, CD-PEI may be a generally useful material for testing novel cell-specific targeting compounds.

BACKGROUND

Peer-assisted learning (PAL) has become a well-accepted teaching method within medical education. However, descriptions of on-ward PAL programmes are rare. We introduced a PAL programme with a focus on clinical competencies on internal medicine wards.

OBJECTIVE

To assess the effects of an on-ward PAL programme on self-assessed clinical competencies.

METHODS

A total of 168 medical students were randomly assigned to one of the seven intervention wards or one of the seven control wards. During their 5-week ward-placement, the intervention group (IG; n = 88) received 10 patient-centred tutorials lead by final year tutors: (I) history taking, (II) physical examination, (III) blood withdrawal, (IV) infusion, (V) patient files, (VI and VII) ECG, (VIII-X) chart rounds. The control group (CG; n = 80) did not take part in the PAL programme. Clinical competencies were self-assessed pre- and post-intervention.

RESULTS

For five of the ten assessed clinical competencies, increases in self-confidence ratings were significantly higher in the IG as compared to CG.

CONCLUSIONS

RESULTS provide preliminary evidence to suggest that PAL programmes on internal medicine wards and with final year students as peer tutors may represent a valuable additional tool within medical clerkships. However, the findings must be confirmed and clarified in further research.

Results are reported for a study of 2 separate processes of report writing-taking notes while reading source material and composing a report from those notes-and related individual differences in executive functions involved in integrating reading and writing during these writing activities. Third graders (n = 122) and 5th graders (n = 106; overall, 127 girls and 114 boys) completed two reading-writing tasks-read paragraph (mock science text)-write notes and use notes to generate written report, a reading comprehension test, a written expression test, four tests of executive functions (inhibition, verbal fluency, planning, switching attention), and a working memory test. For the read-take notes task, the same combination of variables was best (explained the most variance and each variable added unique variance) for 3rd graders and 5th graders: Wechsler Individual Achievement Test-Second Edition (WIAT-II) Reading Comprehension, Process Assessment of the Learner Test for Reading and Writing (PAL) Copy Task B, WIAT-II Written Expression, and Delis-Kaplan Executive Function System (D-KEFS) Inhibition. For the use notes to write report task, the best combinations of variables depended on grade level: For 3rd graders, WIAT-II Reading Comprehension, WIAT-II Written Expression, D-KEFS Verbal Fluency, and Tower of Hanoi; for 5th graders, WIAT-II Reading Comprehension, D-KEFS Verbal Fluency, WIAT-II Written Expression, and PAL Alphabet Task. These results add to prior research findings that executive functions contribute to the writing development of elementary-grade students and additionally support the hypothesis that executive functions play a role in developing reading-writing connections.

Primary adrenal lymphoma (PAL) is extremely uncommon. We describe a case of clinically silent non-Hodgkin's B-cell lymphoma of diffuse large cell type with exclusive left adrenal localization. The tumor was discovered by computed tomography (CT) as a 2.5-cm dense mass and diagnosed at autopsy. Literature concerning this unusual neoplasm is reviewed. During the early stage, particularly when the lesion is small, PAL is likely to be missed. This unusual entity should be included in the differential diagnosis of adrenal masses so that early diagnosis may be made and intervention might dramatically affect the clinical outcome.

1. Evidence from recent experimental and clinical studies suggests that excessive circulating levels of aldosterone can bring about adverse cardiovascular sequelae independent of the effects on blood pressure. Examples of these sequelae are the development of myocardial and vascular fibrosis in uninephrectomized, salt-loaded rats infused with mineralocorticoids and, in humans, an association of aldosterone with left ventricular hypertrophy, impaired diastolic and systolic function, salt and water retention causing aggravation of congestion in patients with established congestive cardiac failure (CCF), reduced vascular compliance and an increased risk of arrhythmias (resulting from intracardiac fibrosis, hypokalaemia, hypomagnesaemia, reduced baroreceptor sensitivity and potentiation of catecholamine effects). 2. These sequelae of aldosterone excess may contribute to the pathogenesis and worsen the prognosis of CCF and hypertension. 3. The heart and blood vessels may be capable of extra-adrenal aldosterone biosynthesis, raising the possibility that aldosterone may have paracrine or autocrine (and not just endocrine) effects on cardiovascular tissues. 4. The high prevalence of CCF, which is associated with secondary aldosteronism, and primary aldosteronism (PAL; recently recognized to be a much more common cause of hypertension than was previously thought) argue for an important role for aldosterone excess as a cause of cardiovascular injury. 5. The recognition of non-blood pressure-dependent adverse sequelae of aldosterone excess raises the question as to whether normotensive individuals with PAL, who have been detected as a result of genetic or biochemical screening among families with inherited forms of PAL, are at excess risk of cardiovascular events. 6. Provided that patients are carefully investigated in order to permit the appropriate selection of specific surgical (laparoscopic adrenalectomy for PAL that lateralizes on adrenal venous sampling) or medical (treatment with aldosterone antagonist medications) management and safety considerations for the use of aldosterone antagonists are kept in mind, the appreciation of a widening role for aldosterone in cardiovascular disease should provide a substantially better outlook for many patients with CCF and hypertension.

Common buckwheat (Fagopyrum esculentum) is a short-season grain crop that is a source of rutin and other phenolic compounds. In this study, we isolated the cDNAs of 11 F. esculentum enzymes in the flavonoid biosynthesis pathway, namely, phenylalanine ammonia lyase (PAL), cinnamate 4-hydroxylase (C4H), 4-coumarate:CoA ligase (4CL) 1 and 2, chalcone synthase (CHS), chalcone isomerase (CHI), flavone 3-hydroxylase (F3H), flavonoid 3'-hydroxylase (F3'H), flavonol synthase (FLS) 1 and 2, and anthocyanidin synthase (ANS). Quantitative real-time polymerase chain reaction analysis showed that these genes were most highly expressed in the stems and roots. However, high performance liquid chromatography analysis indicated that their flavonoid products, such as rutin and catechin, accumulated in the flowers and leaves. These results suggested that flavonoids may be transported within F. esculentum. In addition, light and dark growth conditions affected the expression levels of the biosynthesis genes and accumulation of phenolic compounds in F. esculentum sprouts.

Changes in soluble and cell wall bound peroxidase (POD, EC 1.11.1.7) activity, phenylalanine ammonia-lyase (PAL, EC 4.3.1.5) activity, and lignin content in roots of ferulic acid-stressed soybean (Glycine max (L.) Merr.) seedlings and their relationships with root growth were investigated. Three-day-old soybean seedlings were cultivated in half-strength Hoagland nutrient solution containing 1.0 mM ferulic acid for 24-72 hr. Length, fresh weight, and dry weight of roots decreased, while soluble and cell wall bound POD activity, PAL activity, and lignin content increased after ferulic acid treatment. These enzymes probably participate in root growth reduction in association with cell wall stiffening related to the formation of cross-linking among cell wall polymers and lignin production.

The Rim101/PacC transcription factor acts in a fungal-specific signaling pathway responsible for sensing extracellular pH signals. First characterized in ascomycete fungi such as Aspergillus nidulans and Saccharomyces cerevisiae, the Rim/Pal pathway maintains conserved features among very distantly related fungi, where it coordinates cellular adaptation to alkaline pH signals and micronutrient deprivation. However, it also directs species-specific functions in fungal pathogens such as Cryptococcus neoformans, where it controls surface capsule expression. Moreover, disruption of the Rim pathway central transcription factor, Rim101, results in a strain that causes a hyper-inflammatory response in animal infection models. Using targeted gene deletions, we demonstrate that several genes encoding components of the classical Rim/Pal pathway are present in the C. neoformans genome. Many of these genes are in fact required for Rim101 activation, including members of the ESCRT complex (Vps23 and Snf7), ESCRT-interacting proteins (Rim20 and Rim23), and the predicted Rim13 protease. We demonstrate that in neutral/alkaline pH, Rim23 is recruited to punctate regions on the plasma membrane. This change in Rim23 localization requires upstream ESCRT complex components but does not require other Rim101 proteolysis components, such as Rim20 or Rim13. Using a forward genetics screen, we identified the RRA1 gene encoding a novel membrane protein that is also required for Rim101 protein activation and, like the ESCRT complex, is functionally upstream of Rim23-membrane localization. Homologs of RRA1 are present in other Cryptococcus species as well as other basidiomycetes, but closely related genes are not present in ascomycetes. These findings suggest that major branches of the fungal Kingdom developed different mechanisms to sense and respond to very elemental extracellular signals such as changing pH levels.

The phenylpropanoid pathway yields a variety of phenolics that are closely associated with fruit qualities in addition to structural and defense-related functions. However, very little has been reported concerning its metabolism in fruit. This experiment was designed to assess changes of eleven phenolic acids in grape berry (Vitis vinifera L. cv. Cabernet Sauvignon) and explore both the activities and amounts of three key enzymes--phenylalanine ammonia-lyase (PAL), cinnamate-4-hydroxylase (C4H) and 4-coumarate:coenzyme A ligase (4CL)--catalyzing the biosynthesis of these compounds during berry development. Finally, the subcellular localizations of the enzymes within berry tissues were also investigated using immuno-gold electron microscopic technique. The results indicated that the contents of gallic, protocatechuic, gentisic and caffeic acid all changed drastically during berry development, while other compounds containing p-hydroxybenzoic, vanillic, syringic, chlorogenic, p-coumaric, ferulic and sinapic acid varied only slightly. Activities of PAL, C4H and 4CL showed similar pattern changes with two accumulated peaks throughout berry development. In addition, their activities all showed a highly positive correlation with the total contents of phenolic acids, whereas the immunoblotting analysis showed that changes in enzyme activities were independent of the enzyme amounts. Results from the subcellular-localization study revealed that PAL was mainly present in the cell walls, secondarily thickened walls, and the parenchyma cells of the berry mesocarp cells, C4H was found primarily in the chloroplast (plastid) and nucleus and 4CL predominantly in the secondarily thickened walls and the parenchyma cells of mesocarp vascular tissue.

Production of industrial aromatic chemicals from renewable resources could provide a competitive alternative to traditional chemical synthesis routes. This review describes the engineering of microorganisms for the production of p-hydroxycinnamic acid (pHCA) and p-hydroxystyrene (pHS) from glucose. The initial process concept was demonstrated using a tyrosine-producing Escherichia coli strain that overexpressed both fungal phenylalanine/tyrosine ammonia lyase (PAL) and bacterial pHCA decarboxylase (pdc) genes. Further development of this bioprocess resulted in uncoupling the pHCA and pHS production steps to mitigate their toxicity to the production host. The final process consists of a fermentation step to convert glucose to tyrosine using a tyrosine-overproducing E. coli strain. This step is followed by a single biotransformation reaction to deaminate tyrosine to pHCA through immobilized E. coli cells that overexpress the Rhodotorula glutinis PAL gene. Finally, chemical decarboxylation of pHCA produces pHS. This multifaceted approach, which integrates biology, chemistry, and engineering, has allowed development of an economical process at scales suitable for industrial applications.

The production of alpha-amidated peptides from their glycine-extended precursors is a two-step process involving the sequential action of two catalytic domains encoded by the bifunctional peptidylglycine alpha-amidating monooxygenase (PAM) precursor. The NH2-terminal third of the PAM precursor contains the first enzyme, peptidylglycine alpha-hydroxylating monooxygenase (PHM), a copper, molecular oxygen, and ascorbate-dependent enzyme. The middle third of the PAM precursor contains the second enzyme, peptidyl-alpha-hydroxyglycine alpha-amidating lyase (PAL). The COOH-terminal third of the PAM precursor encodes a transmembrane domain and a hydrophilic domain that may form a cytoplasmic tail. Antisera to a peptide within the PAL domain were used to identify a 50-kDa protein as the major form of PAL in bovine neurointermediate pituitary granules. This 50-kDa PAL protein was purified and found to begin at Asp434 of bPAM, indicating that it could arise through endoproteolytic cleavage of the bPAM precursor at Lys432-Lys433. With alpha-N-acetyl-Tyr-Val-alpha-hydroxyglycine as the substrate, PAL exhibits a pH optimum of 5.0; enzymatic activity is inhibited by high concentrations of salt but is relatively resistant to thiol reagents and urea. PAL activity is inhibited by EDTA and restored by a number of divalent metals, including Cd2+, Cu2+, Zn2+, and Ca2+. Kinetic studies using alpha-N-acetyl-Tyr-Val-alpha-hydroxyglycine indicate that PAL has a Km of 38 microM and a turnover number of 220/s. Expression vectors encoding only the soluble PHM domain or the PAM precursor from which the PHM domain had been deleted were constructed. hEK293 cells transfected with the PHM vector exhibited a 10-fold increase in secretion of PHM activity with no PHM activity detectable in control or transfected cells. hEK293 cells transfected with the PAL vector exhibited a 2-fold increase in secretion of PAL activity and a 15-fold increase in cellular PAL activity. Most of the PAL activity produced by the transfected cells remained membrane-associated.

Phenylalanine ammonia-lyase (<em>PAL</em>) catalyzes the first step in phenylpropanoid metabolism and plays a central role in the biosynthesis of phenylpropanoid compounds. We have previously cloned two <em>PAL</em> genes, <em>PAL</em>I and <em>PAL</em>2, from a Populus trichocarpa x P. deltoides F1 hybrid. Here, we describe the properties of <em>PAL</em>I and <em>PAL</em>2 promoters and their expression patterns in transgenic tobacco and poplar. The promoters were 75% identical in the regions sequenced, and each contained two copies of AC-rich putative cis-acting elements that matched a consensus plant myb transcription factor binding site sequence. In transgenic tobacco, <em>PAL</em>I-GUS and <em>PAL</em>2-GUS fusions directed similar patterns of expression in developing primary xylem of leaves, stems, and other organs, and in secondary xylem of stems. Contrary to previously documented patterns of <em>PAL</em>1/2 expression in poplar, no expression of either fusion was detected in epidermal or subepidermal cell layers of young tobacco leaves or stems. In poplar, the <em>PAL</em>2-GUS fusion directed the highest levels of expression in roots and young leaves and stems. In young leaves and stems, high GUS activity was detected in epidermal or subepidermal cells as well as in primary xylem and phloem fibers. GUS activity was low in woody stems, and was weak or absent in developing secondary xylem. The patterns of <em>PAL</em>2-GUS expression in poplar are very similar to those of <em>PAL</em>1/2 mRNA accumulation in poplar. However, the distinct patterns of expression directed by the <em>PAL</em>2 promoter in poplar and tobacco show that <em>PAL</em>2-GUS expression in tobacco does not accurately reflect all aspects of <em>PAL</em>2 expression in poplar.

Nuclear transcript run-on analysis was used to investigate++ the relative transcription rates of genes encoding enzymes of isoflavonoid phytoalexin biosynthesis and related pathways in elicitor-treated alfalfa (Medicago sativa L.) cell suspension cultures. Genes encoding L-phenylalanine ammonia-lyase (PAL), chalcone synthase (CHS) and chalcone reductase (CHR) were most rapidly activated, with increases in transcription measurable within 10-20 min after elicitation. Cinnamic acid 4-hydroxylase (C4H), chalcone isomerase (CHI), isoflavone reductase (IFR) and caffeic acid 3-0-methyltransferase (COMT) genes were also rapidly activated, but at a slower initial rate. Transcription of chalcone 2'-O-methyltransferase (CHOMT), and 1,3-beta-D-glucanase genes was less rapid, with lag periods of 60 and 30 min post-elicitation, respectively. Treatment of cells with a PAL inhibitor L-alpha-aminooxy-beta-phenylpropionic acid (AOPP) resulted in increased transcription of PAL, CHS and CHR, but reduced transcription of CHOMT, indicating a role for phenylpropanoid products as both negative and positive regulators of gene expression within the phenylpropanoid pathway.

BACKGROUND

Factors influencing the outcome of regenerative therapy of Class II furcations are incompletely and poorly understood. The purpose of this 24-month prospective study was to examine the relationship of patient-, site-, and treatment-related factors to the clinical closure of randomly selected mandibular Class II furcations. Results of therapy were evaluated at 1 and 2 years postoperatively. One-year outcome data are presented in this report.

METHODS

A total of 43 otherwise healthy individuals with chronic periodontitis (26 male, 17 female), 36 to 70 years of age, completed the 12-month evaluation of the study. Entry criteria included clinical and radiographic evidence of two or more mandibular facial Class II furcation defects >> or = 3 mm horizontal probing depth). Surgical therapy was completed by four periodontists (two each) in either a university clinic or private practice. Each patient contributed two furcation defects that were treated by combination therapy using an expanded polytetrafluoroethylene (ePTFE) membrane and demineralized freeze-dried bone allograft (DFDBA). Clinical measurements included a gingival index, plaque index, mobility, and, referencing an occlusal stent, probing depth (PD), probing attachment level-vertical (PAL-V), and probing attachment level-horizontal (PAL-H). Multiple linear measurements were recorded for each site clinically and after surgical debridement to characterize defect morphology, root configuration, and barrier placement. Defect volume was computed mathematically. Postsurgical maintenance care was provided at 1 to 2, 4, 6, and 8 weeks, and then biweekly until 3 months, with subsequent supportive periodontal maintenance visits at 3-month intervals. The clinical status of the furcation (open or closed), measured by a non-treating periodontist at 1 and 2 years, was the primary outcome measure. The association of patient-related factors (e.g., smoking), site-related factors (e.g., root configuration and defect morphology), and treatment-related factors (e.g., membrane exposure) to clinical status of furcations was assessed using random effects hierarchical logistic regression analysis, controlling for design and demographic variables. Non-parametric analysis was used for specific group comparisons.

RESULTS

Complete clinical closure was achieved in 74% of all sites. Of the residual furcation defects, 68% were reduced to Class I. No defects progressed to Class III. Significant improvements in mean PD and PAL-V were obtained following surgical therapy. Although the proportion of sites demonstrating complete furcation closure was comparable for smokers and non-smokers, the proportion of Class II residual defects was significantly higher among smokers than non-smokers (62.5% versus 14.3%, respectively). Increases in presurgical PAL-H were associated with monotonic decreases in the percentage of sites demonstrating complete clinical closure, with only 53% of lesions>> or = 5 mm responding with complete closure. Similarly, significant reductions in the frequency of clinical closure were associated with increases in the distance between the roof of furcation and crest of bone, roof of furcation and base of defect, depth of horizontal defect, and divergence of roots at the crest of bone.

CONCLUSIONS

The successful clinical closure of Class II furcations was achievable at 1 year following combination therapy with an ePTFE membrane and DFDBA. The highest frequency of clinical furcation closure was observed in early Class II defects. Furcations with vertical or horizontal bone loss of 5 mm or greater responded with the lowest frequency of complete clinical closure. Nevertheless, complete furcation closure was achievable in 50% of molars with extensive bone loss. Also, 15 out of 22 (68%) of all residual defects were reduced to Class I and only seven (8%) failed to improve, demonstrating that successful clinical resolution of advanced defects remains an attainable goal.

Phenylalanine ammonia-lyase (PAL) is the first entry enzyme of the phenylpropanoid pathway, and therefore plays a key role in both plant development and stress defense. In many plants, PAL is encoded by a multi-gene family, and each member is differentially regulated in response to environmental stimuli. In the present study, we report that PAL in cucumber (Cucumis sativus L.) is encoded for by a family of seven genes (designated as CsPALPALs are arranged in tandem in two duplication blocks, which are located on chromosomes 4 and 6, respectively. The cDNA and protein sequences of the CsPALs share an overall high identity to each other. Homology modeling reveals similarities in their protein structures, besides several slight differences, implying the different activities in conversion of phenylalanine. Phylogenic analysis places CsPALPALs. Analyses of expression profiles in different cucumber tissues or in response to various stress or plant hormone treatments indicate that CsPALPALs possess overlapping but different cis-elements in their promoter regions. Finally, several duplication events are discussed to explain the evolution of the cucumber PAL genes.

Learning and memory deficits occur in diabetes mellitus. Although the pathogenesis of cognitive impairment in diabetes has not been fully elucidated, factors such as metabolic impairments, vascular complications and oxidative stress are thought to play possible roles. Here we investigated the effect of chronic treatment with vitamin C (50mg/kg, p.o), vitamin E (100mg/kg, p.o) and both together on passive avoidance learning (PAL) and memory in male Wistar control and diabetic rats. Treatments were begun at the onset of hyperglycemia. Passive avoidance learning was assessed 30 days later. Retention was tested 24h after training. At the end, animals were weighed and blood samples were drawn for plasma glucose measurement. Diabetes caused impairment in acquisition and retrieval processes of PAL and memory. The combination of vitamin C and E improved learning and memory in controls and reversed learning and memory deficits in diabetic rats. Combined treatment also affected the body weight and plasma glucose level of diabetic treated animals compared to untreated diabetic animals. Hypoglycemic effects and antioxidant properties of the vitamins may be involved in the nootropic effect of such treatment. These results show that combined treatment with vitamins C and E improved PAL and memory of control rats. In addition, combined vitamins administration to rats for 30 days from onset of diabetes alleviated the negative influence of diabetes on learning and memory. Therefore, combined vitamins treatment may provide a new potential alternative for prevention of impaired cognitive functions associated with diabetes and may warrant further clinical study.