Increasing cleavage specificity and activity of restriction endonuclease KpnI

Restriction enzyme KpnI is a HNH superfamily endonuclease requiring divalent metal ions for DNA cleavage but not for binding. The active site of KpnI can accommodate metal ions of different atomic radii for DNA cleavage. Although Mg2+ ion higher than 500 mu M mediates promiscuous activity, Ca2+ suppresses the promiscuity and induces high cleavage fidelity. Here, we report that a conservative mutation of the metal-coordinating residue D148 to Glu results in the elimination of the Ca2+-mediated cleavage but imparting high cleavage fidelity with Mg2+. High cleavage fidelity of the mutant D148E is achieved through better discrimination of the target site at the binding and cleavage steps. Biochemical experiments and molecular dynamics simulations suggest that the mutation inhibits Ca2+-mediated cleavage activity by altering the geometry of the Ca2+-bound HNH active site. Although the D148E mutant reduces the specific activity of the enzyme, we identified a suppressor mutation that increases the turnover rate to restore the specific activity of the high fidelity mutant to the wild-type level. Our results show that active site plasticity in coordinating different metal ions is related to KpnI promiscuous activity, and tinkering the metal ion coordination is a plausible way to reduce promiscuous activity of metalloenzymes.

Type III restriction-modification enzymes: a historical perspective

Restriction endonucleases interact with DNA at specific sites leading to cleavage of DNA. Bacterial DNA is protected from restriction endonuclease cleavage by modifying the DNA using a DNA methyltransferase. Based on their molecular structure, sequence recognition, cleavage position and cofactor requirements, restriction-modification (R-M) systems are classified into four groups. Type III R-M enzymes need to interact with two separate unmethylated DNA sequences in inversely repeated head-to-head orientations for efficient cleavage to occur at a defined location (25-27 bp downstream of one of the recognition sites). Like the Type I R-M enzymes, Type III R-M enzymes possess a sequence-specific ATPase activity for DNA cleavage. ATP hydrolysis is required for the long-distance communication between the sites before cleavage. Different models, based on 1D diffusion and/or 3D-DNA looping, exist to explain how the long-distance interaction between the two recognition sites takes place. Type III R-M systems are found in most sequenced bacteria. Genome sequencing of many pathogenic bacteria also shows the presence of a number of phase-variable Type III R-M systems, which play a role in virulence. A growing number of these enzymes are being subjected to biochemical and genetic studies, which, when combined with ongoing structural analyses, promise to provide details for mechanisms of DNA recognition and catalysis.

Dietary vitamin E inhibits the trans 10-18:1 shift in beef backfat

[EN]Forty feedlot steers were fed a barleygrain-based finishing diet typical for western Canada, with two levels of supplementary vitamin E (468 or 1068 IU head_1 d_1) and the effect on backfat trans-18:1 isomeric profile was determined. Feeding 1068 IU vitamin E reduced the total trans-18:1 content in backfat (P<0.01), as well as the percentage of trans 10-18:1 (P<0.001), which are related to an increased risk for cardiovascular diseases. On the other hand, trans 11-18:1 (vaccenic acid) the precursor for cis 9,trans 11- 18:2 (rumenic acid), which have several purported health benefits, increased (P<0.01). Vitamin E could, therefore, be used to decrease trans-18:1 in beef and improve its isomeric profile.

The Object Agreement Restriction

[EN]This paper deals with the so-called Person Case Constraint (Bonet, 1991), a universal constraint blocking accusative clitics and object agreement morphemes other than third person when a dative is inserted in the same clitic/agreement cluster. The aim of this paper is twofold. First, we argue that the scope of the PCC is considerably broader than assumed in previous work, and that neither its formulation in terms of person (1st/2nd vs. 3rd)-case (accusative vs. dative) restrictions nor its morphological nature are part of the right descriptive generalization.We present evidence (i) that the PCC is triggered by the presence of an animacy feature in the object’s agreement set; (ii) that it is not case dependent, also showing up in languages that lack dative case; and (iii) that it is not morphologically bound. Second, we argue that the PCC, even if it is modified accordingly, still puts together two different properties of the agreement system that should be set apart: (i) a cross linguistic sensitivity of object agreement to animacy and (ii) a similarly widespread restriction on multiple object agreement observed crosslinguistically. These properties lead us to propose a new generalization, the Object Agreement Constraint (OAC): if the verbal complex encodes object agreement, no other argument can be licensed through verbal agreement.

Food habits and dietary variability of pelagic nekton off Oregon and Washington, 1979-1984

The food habits of 20 species of pelagic nekton were investigated from collections made with small-mesh purse seines from 1979-84 off Washington and Oregon. Four species (spiny dogfish, Squalus acanthias; soupfin shark, Galeorhinus zyopterus; blue shark, Prionace glauca; and cutthroat trout, Salmo clarki) were mainly piscivorous. Six species (coho salmon, Oncorhynchus kisutch; chinook salmon, O. tshawytscha; black rockfish, Sebastes melanops; yellowtail rockfish, S. f1avidus; sablefish, Anoplopoma fimbria; and jack mackerel, Trachurus symmetricus) consumed both nektonic and planktonic organisms. The remaining species (market squid, Loligo opalescens; American shad, Alosa sapidissima; Pacific herring, Clupea harengus pallasi; northern anchovy, Engraulis mordax; pink salmon, O. gorbuscha; surf smelt, Hypomesus pretiosus; Pacific hake, Merluccius productus; Pacific saury, Cololabis saira; Pacific mackerel, Scomber japonicus; and medusafish, Icichthys lockingtom) were primarily planktonic feeders. There were substantial interannual, seasonal, and geographic variations in the diets of several species due primarily to changes in prey availability. Juvenile salmonids were not commonly consumed by this assemblage of fishes (PDF file contains 36 pages.)

Association between dietary phylloquinone intake and peripheral metabolic risk markers related to insulin resistance and diabetes in elderly subjects at high cardiovascular risk

Background: Vitamin K has been related to glucose metabolism, insulin sensitivity and diabetes. Because inflammation underlies all these metabolic conditions, it is plausible that the potential role of vitamin K in glucose metabolism occurs through the modulation of cytokines and related molecules. The purpose of the study was to assess the associations between dietary intake of vitamin K and peripheral adipokines and other metabolic risk markers related to insulin resistance and type 2 diabetes mellitus. Methods: Cross-sectional and longitudinal assessments of these associations in 510 elderly participants recruited in the PREDIMED centers of Reus and Barcelona (Spain). We determined 1-year changes in dietary phylloquinone intake estimated by food frequency questionnaires, serum inflammatory cytokines and other metabolic risk markers. Results: In the cross-sectional analysis at baseline no significant associations were found between dietary phylloquinone intake and the rest of metabolic risk markers evaluated, with exception of a negative association with plasminogen activator inhibitor-1. After 1-year of follow-up, subjects in the upper tertile of changes in dietary phylloquinone intake showed a greater reduction in ghrelin (-15.0%), glucose-dependent insulinotropic peptide (-12.9%), glucagon-like peptide-1 (-17.6%), IL-6 (-27.9%), leptin (-10.3%), TNF (-26.9%) and visfatin (-24.9%) plasma concentrations than those in the lowest tertile (all p<0.05). Conclusion: These results show that dietary phylloquinone intake is associated with an improvement of cytokines and other markers related to insulin resistance and diabetes, thus extending the potential protection by dietary phylloquinone on chronic inflammatory diseases.

The effects of dietary treatment on the morphometrics and haematological characteristics in Clarias gariepinus

Fingerling of Clarias gariepinus have been reported to have an optimal protein requirement of 40%. Not much is known about the effect that varying this protein level has in the haematological characteristics (i.e packed cell volume, red blood cell count, haemoglobin concentration and mean corpuscular haemoglobin concentration) and on some landmarks measured along the fish's body. The haematological parameter are useful in assessing the effect of dietary treatment on leanness or robustness in the fish. The results of these experiment reveals that of the 17 landmarks measured on the bodies of the fish species fed dietary protein levels of no protein 11% (low protein), 29% (sub optimal) and 40% (optimal), only four of the landmarks show significant difference. Also, analysis of the haematological characteristics show significant difference in haematoant (PCV) and erthroycte count (RBC) in all the treatments

Dietary carbohydrate requirement of Heterobranchus longifilis

The dietary carbohydrate requirement of Heterobranchus longifilis was evaluated in two separate experiments.In the first experiment, varying levels of carbohydrate ranging from 28, 24 to58 72% were fed to the fish of mean weight 1.83~c0.02g. Results revealed that the polynomial regression curve for the mean weight gain and the carbohydrate levels did not present a point where Y-max is equal to X-max and so the requirement was not obtained. The second experiment was therefore, conducted with lower levels of carbohydrate ranging from 17.00 to 20.86% and fed to fish with mean weight 0.49~c0.02g. Based on growth and feed efficiency data the carbohydrate requirement was determined to be 19.5%

Growth response of Heterobranchus longifilis fingerlings fed with varying levels of dietary freshwater mussel (Aspatharia sinuata)

A common bivalve: Freshwater mussel, Aspatharia sinuate was evaluated as a dietary protein supplement in the production diet for Heterobranchus longifilis fingerlings with mean body weight 8.34 plus or minus 10g reared in aquaria tanks. Four diets containing fishmeal protein at a rate of 25%, 50% and 75% along with the freshwater mussel flesh were formulated. The diet without the freshwater mussel served as the control. The fish fingerlings were fed at 5% body weight per day for 56 days. It was revealed at the end of the experiment that freshwater mussel was most suitable as a protein supplement when incorporated at 25% replacement. The body weight gain, specific growth rate and feed conversion ration values of 6.83g, 1.06% day and 0.62 respectively were highest in diet with 25% replacement closely followed by diet with 50% replacement. Beyond 75% inclusion level there was no significant growth (P>0.05). However, complete replacement of fishmeal by freshwater mussel decreases growth rates and should not be used in Heterobranchus longifilis diets

Haematological response of Heterobranchus longifilis fed varying dietary protein levels

Hematological effects of feeding varying dietary crude proteins levels to one hundred and fifty (150) H.longifilis fingerlings was examined on biweekly basis. The fingerlings of mean weights 1.26g plus or minus 0.24g were stocked in eight hapa nets (1mx1m) at 15 fingerlings per hapa. Four experimental diets with crude protein; 35%, 40%, 45% and 50% coded diet 1-4 respectively were fed to the fish for 8 weeks. The blood sample was taken and examined for packed cell volume (PCV) total protein (TP) Hemoglobin (Hb), Serum album, Erythrocyte count (RBC), while blood cell (WBC) mean corpuscle volume (MCV) and mean corpuscle hemoglobin, concentration (MCHC). There was an increase in the values of the hematological indices studied with increase in protein inclusion levels. A higher positive correlation with no significant difference (P greater than or equal to 0.05) exists between the treatments RBC, WBC, Hb and TP. The best RBC (2.10x10 super(6) count/l). WBC (7.65x10 super(4) count/l), PCV (35.4%) and Hb (5.79mg/l) were presented in fingerlings fed 40% crude protein followed by 45% crude protein. The dietary crude protein of 40% is recommended for H. longifilis for sound and healthy condition

Flood circulation in oligomictic lakes: an undervalued reason for restriction of zooplankton production [Translation from: Memorie dell'Istituto Italiano di Idrobiologia Dott.Marco de Marchi 22 9-52, 1967]

Dormancy has been observed in several species of Cyclops. Animals in dormancy are characterised by almost complete inactivity of the main appendages and sluggish intestinal peristalsis. In Cyclops vicinus dormancy can take place in copepod stages III (19%), IV (78%) and V (3%) but one and the same animal only in one developmental stage. The author gives his own results between frequency of dormancy and photoperiod (20 C, 1000 lux). He concludes that dormancy in C. vicinus can be influenced by day length and can be ended prematurely by short day length.

Análise da concordância entre o plano dietético Dietary Approaches to Stop Hypertension (DASH) e o padrão alimentar de pacientes hipertensos

A adoção do plano dietético Dietary Approaches to Stop Hypertension (DASH) tem sido enfatizado na população hipertensa como uma importante estratégia no controle dos níveis pressóricos elevados. O objetivo deste estudo foi analisar o consumo alimentar de macro e micronutrientes em pacientes hipertensos, em especial do sódio, cálcio, potássio e magnésio, e comparar com as recomendações dietéticas contidas no plano DASH. Estudo transversal envolvendo uma amostra de conveniência composta por 113 hipertensos entre 40 e 69 anos. A medida da pressão arterial (PA) foi determinada com aparelho eletrônico devidamente calibrado e a avaliação dietética obtida pelo questionário de freqüência do consumo alimentar. Os alimentos ingeridos foram convertidos em porções e distribuídos em diferentes grupos alimentares. A faixa de porções recomendadas pelo plano DASH foi determinada com base na média das necessidades energéticas desta amostra permitindo assim uma posterior comparação com o hábito alimentar dos hipertensos, utilizando um escore de pontos com pontuação máxima de 9 pontos. A amostra foi dividida em três grupos: grupo B que obteve 2,5 a 4,0 pontos (n=34; 30%), grupo M entre 4,5 a 5,0 pontos (n=43; 38%) e grupo A que obteve 5,5 a 8,0 pontos (n=36; 32%). Não foram observadas diferenças significativas na média da PA sistólica (14024 vs 13823 vs 13515 mmHg) e diastólica (8617 vs 8813 vs 8410 mmHg). Apesar do grupo A consumir mais proteínas e gordura monoinsaturada, foi detectado um excesso pronunciado na ingestão de gordura saturada, colesterol e das calorias totais, por este grupo de pacientes. No que tange a ingestão dos micronutrientes, o grupo A apresentou consumo significativamente maior de cálcio, potássio e magnésio, refletido pela maior ingestão de vegetais e frutas em comparação aos grupos B e M. A média de ingestão do sódio intrínseco foi significativamente maior no grupo A (4,12,0 vs 3,11,1 vs 2,71,1 g/dia). Foram detectadas, apenas no grupo A, correlações entre PA sistólica e o percentual de proteína (r = -0,5; p=0,002) e PA sistólica e o percentual de carboidrato (r = 0,4; p=0,02). Apenas um terço dos hipertensos avaliados apresentaram padrão alimentar mais concordante com o plano DASH e com maior ingestão de proteínas, gordura monoinsaturada, fibras, cálcio, potássio e magnésio. Entretanto, o consumo mais elevado de sódio, gordura saturada, colesterol e das calorias totais por este grupo de pacientes poderia restringir uma maior queda dos níveis pressóricos elevados.

Dietary variations in three co-occurring rockfish species off the Pacific Northwest during anomalous oceanographic events in 1998 and 1999

Stomach samples from three rockfish species, yellowtail (Sebastes f lavidus), widow (S. entomelas), and canary (S. pinniger) rockfish, seasonally collected off the Pacific Northwest in 1998 and 1999, provided quantitative information on the food habits of these species during and after the 1997–98 El Niño event. Although euphausiids were the most common major prey of all three predators, gelatinous zooplankton and fishes were the most commonly consumed prey items during some seasonal quarters. The influence of the El Niño event was evident in the diets. Anomalous prey items, including the southern euphausiid species Nyctiphanes simplex and juveniles of Pacific whiting (Merluccius productus) frequently appeared in the diets in the spring and summer of 1998. The results of stomach contents analyses, based on 905 stomach samples from 49 trawl hauls during seven commercial fishing trips and from 56 stations during research surveys, were consistent with the timing of occurrence and the magnitude of change in biomass of some zooplankton species reported from zooplankton studies in the northern California Current during the 1997–98 El Niño. Our findings indicate that the observed variations of prey groups in some rockfish diets may be a function of prey variability related to climate and environment changes.

Comparing the identification of southern California juvenile rockfishes (genus Sebastes spp.) by restriction site analysis of the mitochondrial ND3/ND4 region and by morphological characteristics

Rockfish (Sebastes spp.) juveniles are often difficult to identify by using morphological characters. This study independently applies morphological characters and a key based on mitochondrial restriction site variation to identify juvenile rockf ishes collected in southern California during juvenile rockfish surveys. Twenty-four specimens of Sebastes were examined genetically without knowledge of the morphological assignment. Seventeen fish were identified genetically as S. semicinctus, S. goodei, S. auriculatus, S. jordani, S. levis, S. rastrelliger, and S. saxicola. Identities for the remaining fish were narrowed to two or three species: 1) three fish were either S. carnatus or S. chrysomelas; 2) one fish was either S. chlorosticus, S. eos, or S. rosenblatti; and 3) three fish could have been either S. hopkinsi or S. ovalis, the latter for which we now have distinguishing mitochondrial markers. The genetic and morphological assignments concurred except for the identity of one fish that could only be narrowed down to S. hopkinsi or S. semicinctus by using morphological characters. Genetics excluded more species from multispecies groupings than did the morphological approach, especially species within the subgenus Sebastomus. Species in the genetically unresolvable groups may be similar because of recent divergence or because of interspecies introgression.