Behavioral characterization of mice lacking the ubiquitin ligase UBR1 of the N-end rule pathway

The N-end rule pathway, a subset of the ubiquitin pathway, relates the in vivo half-life of a protein to the identity of its N-terminal residue. Mice lacking NTAN1, a component of the N-end rule pathway, showed altered learning and memory, and socially conditioned behavioral alteration ( Balogh, Kwon, & Denenberg 1999, 2000 ; Kwon, Balogh et al . 2000 ; Balogh et al . 2001 ). Mice lacking UBR1 (E3α), one of at least three recognition components of the N-end rule pathway, are viable and outwardly normal. Here we describe behavioral characterizations of UBR1 knockout (UBR1^–/–) mice. Compared to congenic littermates, UBR1^–/– mice exhibited less spontaneous activity in an open field and took longer to locate the hidden platform during eight-week Morris water maze retention. In contrast, they performed better in horizontal–vertical discrimination and Lashley III maze testing. No statistically significant differences in inhibitory learning were observed. With the exception of enhanced Lashley III maze performance, these data parallel findings with NTAN1^–/– mice lacking the upstream component of UBR1. These results suggest that, like NTAN1, UBR1 is involved in learning and memory.     

Multidrug resistance gene deficient (mdr1a–/–) mice have an altered caecal microbiota that precedes the onset of intestinal inflammation

Aim:  To compare caecal microbiota from mdr1a ^–/– and wild type (FVB) mice to identify differences in the bacterial community that could influence the intestinal inflammation.
Methods and Results:  Caecal microbiota of mdr1a ^–/– and FVB mice were evaluated at 12 and 25 weeks of age using denaturing gradient gel electrophoresis (DGGE) and quantitative real-time PCR. DGGE fingerprints of FVB and mdr1a ^–/– mice (with no intestinal inflammation) at 12 weeks revealed differences in the presence of DNA fragments identified as Bacteroides fragilis , B. thetaiotaomicron , B. vulgatus and an uncultured alphaproteobacterium. Escherichia coli and Acinetobacter sp. were only identified in DGGE profiles of mdr1a ^–/– mice at 25 weeks (with severe intestinal inflammation), which also had a lower number of total bacteria in the caecum compared with FVB mice at same age.
Conclusions:  Differences found in the caecal microbiota of FVB and mdr1a ^–/– mice (12 weeks) suggest that the lack of Abcb1 transporters in intestinal cells due to the disruption of the mdr1a gene might lead to changes in the caecal microbiota. The altered microbiota along with the genetic defect could contribute to the development of intestinal inflammation in mdr1a ^–/– mice.
Significance and Impact of the Study:  Differences in caecal microbiota of mdr1a ^–/– and FVB mice (12 weeks) suggest genotype specific colonization. The results provide evidence that Abcb1 transporters may regulate host interactions with commensal bacteria. Future work is needed to identify the mechanisms involved in this possible cross-talk between the host intestinal cells and microbiota.     

Resting and arecoline-stimulated brain metabolism and signaling involving arachidonic acid are altered in the cyclooxygenase-2 knockout mouse

Studies were performed to determine if cyclooxygenase (COX)-2 regulates muscarinic receptor-initiated signaling involving brain phospholipase A₂ (PLA₂) activation and arachidonic acid (AA; 20 : 4n-6) release. AA incorporation coefficients, k* (brain [1–¹⁴C]AA radioactivity/integrated plasma radioactivity), representing this signaling, were measured following the intravenous injection of [1–¹⁴C]AA using quantitative autoradiography, in each of 81 brain regions in unanesthetized COX-2 knockout (COX-2^–/–) and wild-type (COX-2^+/+) mice. Mice were administered arecoline (30 mg/kg i.p.), a non-specific muscarinic receptor agonist, or saline i.p. (baseline control). At baseline, COX-2^–/– compared with COX-2^+/+ mice had widespread and significant elevations of k*. Arecoline increased k* significantly in COX-2^+/+ mice compared with saline controls in 72 of 81 brain regions, but had no significant effect on k* in any region in COX-2^–/– mice. These findings, when related to net incorporation rates of AA from brain into plasma, demonstrate enhanced baseline brain metabolic loss of AA in COX-2^–/– compared with COX-2^+/+ mice, and an absence of a normal k* response to muscarinic receptor activation. This response likely reflects selective COX-2-mediated conversion of PLA₂-released AA to prostanoids.     

Exaggerated emotional behavior in mice heterozygous null for the sodium channel Scn8a (Nav1.6)

The Scn8a gene encodes the α-subunit of Na_v1.6, a neuronal voltage-gated sodium channel. Mice homozygous for mutations in the Scn8a gene exhibit motor impairments. Recently, we described a human family with a heterozygous protein truncation mutation in SCN8A . Rather than motor impairment, neuropsychological abnormalities were more common, suggesting a role for Scn8a in a more diverse range of behaviors. Here, we characterize mice heterozygous for a null mutation of Scn8a ( Scn8a^+/− mice) in a number of behavioral paradigms. We show that Scn8a^+/− mice exhibit greater conditioned freezing in the Pavlovian fear conditioning paradigm but no apparent abnormalities in other learning and memory paradigms including the Morris water maze and conditioned taste avoidance paradigm. Furthermore, we find that Scn8a^+/− mice exhibit more pronounced avoidance of well-lit, open environments as well as more stress-induced coping behavior. Together, these data suggest that Scn8a plays a critical role in emotional behavior in mice. Although the behavioral phenotype observed in the Scn8a^+/− mice only partially models the abnormalities in the human family, we anticipate that the Scn8a^+/− mice will serve as a valuable tool for understanding the biological basis of emotion and the human diseases in which abnormal emotional behavior is a primary component.     

Studies on the toxicity of cadmium to the three-spined stickleback Gasterosteus aculeatus L.

Cadmium was found to be lethal to sticklebacks at all concentrations from 100.0 to 0.001 mg Cd 1^–1, in water of 103–111 mg 1^–1 hardness as CaCO₃. The pattern of mortality as shown by the time-concentration curve suggests that toxicity is not due to a single mechanism but changes with concentration. Fish were found to accumulate cadmium, the whole body levels increasing from 0.90 μg/g fresh weight at 0.001 mg Cd 1^–1 exposure concentration to 51.0 μg/g at 100 mg Cd 1^–1. The concentration factor was shown to decrease with increasing exposure concentration from 0.51 at 100 mg Cd 1^–1 to 511 at 0.001 mg Cd 1^–1. The plerocercoid parasite Schistocephalus solidus in the host's perivisceral cavity contained less cadmium than the tissues of its host.     

The standard metabolic rate of dolphin fish

The standard metabolic rates (SMRs) of 11 (1.395–4.125 kg) dolphin fish (mahimahi or dorado, Coryphaena hippurus ) were measured at 25°± 0.5°C. Fish were prevented from swimming with neuromuscular blocking agents and force ventilated. Heart rates were determined simultaneously. SMRs (358–726 mg O₂ h ^–1) were several times those of other similarly sized active teleosts such as salmonids, but close to those of tunas. Heart rates (84–161 beats min ^–1) were also high, but alike those of tunas under similar circumstances. As in tunas, the high SMR of dolphin fish may result from high osmoregulatory costs engendered by their large gill surface areas and/or other adaptations necessary for achieving exceptionally high maximum metabolic rates.     

Early development of milkfish: effects of salinity on embryonic and larval metabolism, yolk absorption and growth

During embryogenesis of Chanos chanos , more than half of the yolk was consumed and the majority of it was converted into larval tissue. Salinity affected both yolk absorption and embryonic and larval growth. Larvae hatched in 20% had larger yolk reserves but were smaller and grew more slowly than larvae in 35 and 50%. Larvae hatched in 35 and 50% had equal amounts of yolk but those from 35% were larger. Oxygen consumption rates increased during development (from 0.06 ± 0.01 μl O₂ egg^–1 h^–1 by blastulae to 0.37 ± 0-01 μl O₂ egg^–1 h^–1 by prehatch embryos and 0–43 ± 0–03 μl O₂ larva ^–1 h ^–1 by newly-hatched larvae) and were significantly affected by salinity. Eggs and yolk-sac larvae incubated in 35% consumed more oxygen than those in the low and high salinities. Salinity affected both the rate and pattern of yolk utilization but salinity-related differences in metabolism, yolk absorption, and growth were not related directly to the osmotic gradient. Low salinity retarded yolk absorption while high salinity reduced yolk utilization efficiencies. Differences in oxygen consumption rates were probably related to variations in the relative amounts of metabolically active embryonic and larval tissue and/or higher activity levels rather than differential osmoregulatory costs. 35% is probably the most suitable salinity for incubation and larval rearing of milkfish.     

Brain Glutamate Decarboxylase: Properties of Its Calcium-Dependent Binding to Liposomes and Kinetics of the Bound and the Free Enzyme

Abstract: In the present work we describe several properties of the Ca²⁺-dependent binding of glutamate decarboxylase (GAD) to phosphatidylcholine-phosphatidylserine liposomes. The binding occurs very rapidly, is dependent on temperature in the range 23–37°C, is inhibited up to 35% by K⁺ in a concentration-dependent manner and is slightly increased when the dielectric constant of the medium is decreased by 3% ethanol. The association of GAD and liposomes is very firm, since EGTA displaces only 40% of the bound enzyme and Triton X-100 about 55%. Since apparently only part of the total GAD is able to bind to the liposomes and in a previous study two forms of GAD activity have been identified kinetically, we compared the activations by pyridoxal 5'-phosphate (PLP) of the soluble and the bound GAD, as well as their inhibition by PLP oxime- O -acetic acid. The bound GAD was activated 150–265% by 10⁻⁶ to 10⁻⁴ m -PLP, whereas the activation of GAD that remained soluble was only 65–110% in the same PLP concentration range. In the absence of PLP, the bound GAD was less inhibited by the PLP oxime- O -acetic acid than the soluble GAD, but the inhibition was similar when 0.1 m m -PLP was added. In contrast, activity of both the soluble and the bound GAD was totally blocked by aminooxyacetic acid. Endogenous PLP did not bind to liposomes under the experimental conditions inducing GAD binding. We conclude that the binding of GAD to negatively charged liposomes is primarily ionic. Furthermore, the GAD molecules that bind to the liposomes seem to be deficient in free PLP and therefore, are probably more susceptible to regulation by the coenzyme. These conclusions may be relevant to the hypothesis of a coupling between synthesis and release of GABA in inhibitory nerve endings.     

Effect of chelating agents on bud induction and accumulation of iron and copper by the moss Bartramidula bartramioides

The chelating agents, EDDHA, its iron salt, EDTA, and salicylic acid enhance bud formation in Bartramidula bartramioides (Griff.) Wijk & Marg. Salicylic acid elicits optimal response at 10^–4 M , whereas the other substances do so at 10^–7 M . Increased concentration of ferric citrate and cupric sulphate also stimulate bud induction. The accumulation of Fe³⁺ and Cu²⁺ is facilitated by chelators. The endogenous iron content is maximum at 10^–7 M EDDHA or EDTA, which is also the concentration optimal for bud induction.     

Seasonal patterns of nitrogen fixation in termites

1. Termite nitrogenase activity was highest in autumn and spring (≈ 3 μg N₂ fixed termite fresh mass (g)^–1 day^–1) and lowest in winter and summer (≈ 0·8 μg N₂ fixed termite fresh mass (g)^–1 day^–1).
2. The nitrogenase activity of worker termites was significantly higher than all other castes (1·58 ± 0·27 μg N₂ fixed termite fresh mass (g)^–1 day^–1).
3. Worker termites constituted the largest proportion of all the castes throughout the study period (≈ 90%).
4. The localized input of fixed nitrogen by termites may reach 15·3 mg N log^–1 day^–1 and 5·6 g N log^–1 year^–1.     

Aged endothelial nitric oxide synthase knockout mice exhibit higher mortality concomitant with impaired open-field habituation and alterations in forebrain neurotransmitter levels

Endothelial nitric oxide synthase (eNOS) has been implicated in various brain and peripheral pathologies such as renal failure, heart failure or stroke. Consequently, the mortality rate of aged eNOS knockout mice (eNOS^–/–) was higher than that of age-matched (18–22 months old) controls. Only seven of the original 14 eNOS^–/– animals that participated in the study reached the age of 18 months or older, whereas no control mice died during this life span. In order to assess the behavioral and neurochemical consequences of chronic eNOS deficiency we examined whether the surviving aged eNOS^–/– mice showed changes in terms of motor, emotional, exploratory and neurochemical parameters. Aged eNOS^–/– mice showed reduced exploratory activity in the open-field with no habituation observable neither within sessions nor after repeated exposures. Pole test performance of eNOS^–/– mice was comparable to controls. In the elevated plus-maze eNOS^–/– mice did not differ from controls in terms of time spent in and entries into arms, but showed less locomotion on the open arms. The most prominent neurochemical alterations in the forebrains of aged eNOS^–/– mice were: (a) increased acetylcholine levels in the neostriatum; (b) decreased noradrenaline concentrations in the ventral striatum; and (c) lower serotonin levels in the frontal cortex and ventral striatum. The present findings suggest that mice which survived chronic eNOS-deficiency into old age, show some behavioral and neurochemical phenotypes distinct from adult eNOS^–/– mice.     

Expression of freezing and fear‐potentiated startle during sustained fear in mice

Fear‐potentiated acoustic startle paradigms have been used to investigate phasic and sustained components of conditioned fear in rats and humans. This study describes a novel training protocol to assess phasic and sustained fear in freely behaving C57BL/6J mice, using freezing and/or fear‐potentiated startle as measures of fear, thereby, if needed, allowing in vivo application of various techniques, such as optogenetics, electrophysiology and pharmacological intervention, in freely behaving animals. An auditory Pavlovian fear conditioning paradigm, with pseudo‐randomized conditioned–unconditioned stimulus presentations at various durations, is combined with repetitive brief auditory white noise burst presentations during fear memory retrieval 24 h after fear conditioning. Major findings are that (1) a motion sensitive platform built on mechano‐electrical transducers enables measurement of startle responses in freely behaving mice, (2) absence or presence of startle stimuli during retrieval as well as unpredictability of a given threat determine phasic and sustained fear response profiles and (3) both freezing and startle responses indicate phasic and sustained components of behavioral fear, with sustained freezing reflecting unpredictability of conditioned stimulus (CS)/unconditioned stimulus (US) pairings. This paradigm and available genetically modified mouse lines will pave the way for investigation of the molecular and neural mechanisms relating to the transition from phasic to sustained fear.     

Estimating the cost of flowering in a grapefruit tree

The objective of the present study is to evaluate a Citrus tree's investment in the flowering process in relation to its photoassimilate resources, as a part of its annual reproductive effort. The overall requirement for carbohydrate of a single flower of grapefruit ( Citrus paradisi Macf. cv. 'Marsh seedless') is evaluated as 8·33 × 10^–3 mol C over 3 weeks. The direct cost of production of a single flower is estimated to be 5·75 × 10^–3 mol C, most of which is allocated to the petals, anthers and style — organs designated to abscise. About 2·58 × 10^–3 mol C is consumed by respiration not associated with growth processes. Growth respiration ( R _g) occurs mostly during early stages of flower growth and development. However, the total respiration rate increases sharply during anthesis, when growth processes have almost ceased. Ethylene evolution also reaches remarkably high rates during anthesis. High temperatures increase the rate of flower respiration ( Q ₁₀ = 2·12) but shorten the duration of flowering. A grapefruit tree may bear each year 20 000–50 000 flowers, only 0·5–2·5% of which develop into mature fruit. The amount of carbohydrate invested each year in bloom at the whole-tree level is 166–400 mol C per tree (depending on the number of flowers), amounting to 10–20% of the carbohydrate consumed for fruit growth. The overall daily demand for carbohydrate by the flowers of a grapefruit tree during anthesis may exceed the daily carbohydrate production by the leaves. High temperatures lead to a further increase in the daily demand for carbohydrate. In such cases, the management of flowering must rely on carbohydrate reserves recruited from other tree organs. The ecophysiological and evolutionary aspects of Citrus flowering are discussed.     

EFFECTS OF DEPOLARIZATION ON COFACTOR REGULATION OF GLUTAMIC ACID DECARBOXYLASE IN SUBSTANTIA NIGRA SYNAPTOSOMES

Abstract— The level of saturation of glutamate decarboxylase (GAD) by cofactor, pyridoxal-5'-phosphate (pyridoxal-P), determined in synaptosomes prepared from substantia nigra tissue, was reduced from 45 to 28%; when ATP was included in the homogenizing medium to prevent nonspecific activation of GAD by endogenous pyridoxal-P. When the synaptosomes were incubated for 5–20 min at 37°C in Krebs-Ringer phosphate buffer (KRP), the level of saturation of GAD by cofactor decreased further, from 28 to 20%. Depolarization of the nigral synaptosomes by either high K⁺ (55 mM) or veratridine resulted in a significant increase in the level of GAD saturation by cofactor, to 32 and 41%. respectively. Omitting Ca²⁺ from the incubation medium blocked the depolarization-induced rise in the level of saturation. Depolarization with high K⁺ and veratridine also caused a significant decrease in the ATP concentration in the synaptosomes. No difference in ATP concentration was observed when the samples were incubated at 37°C for 5–20min or incubated in the absence of added Ca²⁺ with high K⁺. Results provide further evidence that in vivo brain GAD is largely unsaturated by cofactor and support the possibility that increased release and utilization of GABA may be associated with increases in the amount of pyridoxal-P endogenously bound to GAD in nerve terminals.     

Physiological mechanisms of buoyancy in eggs from brackish water cod

Newly fertilized eggs of brackish water (Gotland, Baltic Sea) and marine (Lofoten, Norway) cod were investigated with regard to specific gravity, wet and dry weight, water content, chorion weight, and content of protein, free amino acids (FAA), and ions. The eggs had neutral buoyancies equivalent to a salinity of 14^.3% (range 11^.5–16^.2%) in brackish water, and 33^.0% (range 31^.8–34^.5%) in the marine environment. A buoyancy model was developed and showed that this difference was mainly caused by differences in egg water content which was 96.6 ± 0^.47% and 92.7 ± 0.45% in the brackish and marine eggs, respectively. The higher water content of the brackish eggs resulted from increased water uptake during final oocyte maturation due to higher intracellular contents of FAA, Cl ^– and NH₄⁺. SDS polyacrylamide gel electrophoresis of eggs and oocytes, and measurements of egg protein content suggested that the FAA pool of both egg types originated from hydrolysis of specific yolk proteins. The main contributor seemed to be a protein with a molecular weight of 100 kDa.     

Actual escape area and lateral escape from the xylem of the alkali ions Na+, K+, Rb+ and Cs+ in tomato

Approximation of the total escape area of the xylem in an inbred line of tomato (Ly-copersicon escutentum Mill. cv. Tiny Tim) with help of the frequency distribution of xylem vessel radii provides the possibility to calculate realistic escape constant values from uptake experiments of several elements into tomato stem segments. Comparison of the lateral escape rates of ²⁴Na⁺, ⁴²K⁺, ⁸⁶Rb⁺ and ¹³⁴Cs⁺ indicate that Na⁺ escape is rate-limited by its uptake into a rather constant number of surrounding cells, regardless of changes in the total escape area of the xylem vessels. The escape of K⁺, Rb⁺ and Cs⁺ seems to be proportional to the surface area of the xylem vessels and their escape is apparently controlled by their transport across the cell walls of the transport channels. The calculated small values for the escape rate constants (apparent permeability of the xylem cell walls, ca 2–3 · 10−⁹ m s−⁷) are probably due to the presence of lignin in the xylem cell walls, the discrimination between ions as a result of differing affinities and selectivities and the presence of other solutes in the applied solution.     

Reduced microbial diversity and high numbers of one single Escherichia coli strain in the intestine of colitic mice

Commensal bacteria play a role in the aetiology of inflammatory bowel diseases (IBD). High intestinal numbers of Escherichia coli in IBD patients suggest a role of this organism in the initiation or progression of chronic gut inflammation. In addition, some E. coli genotypes are more frequently detected in IBD patients than others. We aimed to find out whether gut inflammation in an IBD mouse model is associated with a particular E. coli strain. Intestinal contents and tissue material were taken from 1-, 8-, 16- and 24-week-old interleukin 10-deficient (IL-10^–/–) mice and the respective wild-type animals. Caecal and colonic inflammation was observed in IL-10^–/– animals from the 8 weeks of life on accompanied by a lower intestinal microbial diversity than in the respective wild-type animals. Culture- based and molecular approaches revealed that animals with gut inflammation harboured significantly higher numbers of E. coli than healthy controls. Phylogenetic grouping according to the E. coli Reference Collection (ECOR) system and strain typing by random-amplified polymorphic DNA and pulsed-field gel electrophoresis revealed that all mice were colonized by one single E. coli strain. The strain was shown to have the O7:H7:K1 serotype and to belong to the virulence-associated phylogenetic group B2. In a co-association experiment with gnotobiotic mice, the strain outnumbered E. coli ECOR strains belonging to the phylogenetic group A and B2 respectively. A high number of virulence- and fitness-associated genes were detected in the strain's genome possibly involved in the bacterial adaptation to the murine intestine.     

Postnatal handling reverses social anxiety in serotonin receptor 1A knockout mice

Mice lacking the serotonin receptor 1A (Htr1a knockout, Htr1a ^KO ) show increased innate and conditioned anxiety. This phenotype depends on functional receptor activity during the third through fifth weeks of life and thus appears to be the result of long-term changes in brain function as a consequence of an early deficit in serotonin signaling. To evaluate whether this phenotype can be influenced by early environmental factors, we subjected Htr1a knockout mice to postnatal handling, a procedure known to reduce anxiety-like behavior and stress responses in adulthood. Offspring of heterozygous Htr1a knockout mice were separated from their mother and exposed 15 min each day from postnatal day 1 (PD1) to PD14 to clean bedding. Control animals were left undisturbed. Maternal behavior was observed during the first 13 days of life. Adult male offspring were tested in the open field, social approach and resident–intruder tests and assessed for corticosterone response to restraint stress. Knockout mice showed increased anxiety in the open field and in the social approach test as well as an enhanced corticosterone response to stress. However, while no effect of postnatal handling was seen in wild-type mice, handling reduced anxiety-like behavior in the social interaction test and the corticosterone response to stress in knockout mice. These findings extend the anxiety phenotype of Htr1a ^KO mice to include social anxiety and demonstrate that this phenotype can be moderated by early environmental factors.     

Effect of plant growth hormones and polyamines on ornithine decarboxylase activity during the germination of barley seeds

Gibberellic acid (GA₃) and β-indolylacetic acid (IAA), two of the well known growth hormones, induce four fold the activity of ornithine decarboxylase (ODC) during the germination of barley seeds ( Hordeum vulgare L. var. Beca). The optimal concentration for induction of ODC was 10^–5 M for GA₃ and 10^–3 M for IAA. When 10^–3 M of a polyamine, putrescine or spermidine, is added to the growth medium, ODC activity is significantly inhibited. This inhibition is due to the induction of a protein inhibitor of ODC (antizyme), whose apparent molecular weight is 16 000 ± 2 000 daltons. Addition of GA₃ to cultures which have been grown for 50 or 98 h in the presence of polyamines, abolishes the observed inhibition of ODC activity, while in the reverse experiment, addition of polyamines at 50 or 98 h does not affect the ODC activity induced by GA₃. Cadaverine, a physiological plant diamine, enhances ODC activity; whereas 1,8-diaminooctane (the alkyl analogue of spermidine) does not have any effect.     

Effects of Increased γ-Aminobutyric Acid Levels on GAD67 Protein and mRNA Levels in Rat Cerebral Cortex

Abstract: Rats were injected with saline or the γ-aminobutyric acid (GABA) transaminase inhibitor γ-vinyl-GABA for 7 days and the effects on GABA content and glutamic acid decarboxylase (GAD) activity, and the protein and mRNA levels of the two forms of GAD (GAD₆₇ and GAD₆₅) in the cerebral cortex were studied. γ-Vinyl-GABA induced a 2.3-fold increase in GABA content, whereas total GAD activity decreased by 30%. Quantitative immunoblotting showed that the decline in GAD activity was attributable to a 75–80% decrease in GAD₆₇ levels, whereas the levels of GAD₆₅ remained unchanged. RNA slot-blotting with a ³²P-labeled GAD₆₇ cDNA probe demonstrated that the change in GAD₆₇ protein content was not associated with a change in GAD₆₇ mRNA levels. Our results suggest that GABA specifically controls the level of GAD₆₇ protein. This effect may be mediated by a decreased translation of the GAD₆₇ mRNA and/or a change in the stability of the GAD₆₇ protein.