Identification of efflux proteins using efficient radial basis function networks with position‐specific scoring matrices and biochemical properties

Efflux proteins are membrane proteins, which are involved in the transportation of multidrugs. The annotation of efflux proteins in genomic sequences would aid to understand the function. Although the percentage of membrane proteins in genomes is estimated to be 25–30%, there is no information about the content of efflux proteins. For annotating such class of proteins it is necessary to develop a reliable method to identify efflux proteins from amino acid sequence information. In this work, we have developed a method based on radial basis function networks using position specific scoring matrices (PSSM) and amino acid properties. We noticed that the C‐terminal domain of efflux proteins contain vital information for discrimination. Our method showed an accuracy of 78 and 92% in discriminating efflux proteins from transporters and membrane proteins, respectively using fivefold cross‐validation. We utilized our method for annotating the genomes E. coli and P. aeruginosa and it predicted 8.7 and 9.2% of proteins as efflux proteins in these genomes, respectively. The predicted efflux proteins have been compared with available experimental data and we observed a very good agreement between them. Further, we developed a web server for classifying efflux proteins and it is freely available at http://rbf.bioinfo.tw/～sachen/EFFLUXpredict/Efflux‐RBF.php . We suggest that our method could be an effective tool for annotating efflux proteins in genomic sequences.Proteins 2013. © 2013 Wiley Periodicals, Inc.     

The two‐phase partitioning system – a powerful technique to purify integral membrane proteins of Corynebacterium glutamicum for quantitative shotgun analysis

We established a single consecutive strategy which assigned the most comprehensive number of integral membrane proteins from Gram‐positive bacteria to date. For this purpose, we adapted a biphasic partitioning system for the biotechnologically intensively used Corynebacterium glutamicum and proved for the first time that such a system is well suited for quantitative comparison. 297 integral membrane proteins were identified by our integrated approach, which depletes stringently cytosolic proteins. In combination with our previously developed SIMPLE strategy, our data comprise 61% (374 integral membrane proteins) of the entire membrane proteome, which aims towards an almost comprehensive coverage. Wild type and a production strain of C. glutamicum were compared by ¹⁵N metabolic labelling and quantitation was obtained by spectral counting and peak areas. Both quantification strategies display a consistent trend in up or downregulation of proteins. Nevertheless, spectral counting often provides results indicating a much stronger regulation compared to ProRata values. Either spectral counting seems to exaggerate protein regulation or ProRata tends to attenuate the information about the regulation level. We highlight some of the biologically relevant candidates, which prove that our approach helps to give a deeper quantitative insight towards the understanding of transport and other membrane associated processes, important for strain development of C. glutamicum.     

Intrinsic and extrinsic uncoupling of oxidative phosphorylation

This article reviews parameters of extrinsic uncoupling of oxidative phosphorylation (OxPhos) in mitochondria, based on induction of a proton leak across the inner membrane. The effects of classical uncouplers, fatty acids, uncoupling proteins (UCP1-UCP5) and thyroid hormones on the efficiency of OxPhos are described. Furthermore, the present knowledge on intrinsic uncoupling of cytochrome c oxidase (decrease of H⁺/e⁻ stoichiometry=slip) is reviewed. Among the three proton pumps of the respiratory chain of mitochondria and bacteria, only cytochrome c oxidase is known to exhibit a slip of proton pumping. Intrinsic uncoupling was shown after chemical modification, by site-directed mutagenesis of the bacterial enzyme, at high membrane potential ΔΨ, and in a tissue-specific manner to increase thermogenesis in heart and skeletal muscle by high ATP/ADP ratios, and in non-skeletal muscle tissues by palmitate. In addition, two mechanisms of respiratory control are described. The first occurs through the membrane potential ΔΨ and maintains high ΔΨ values (150-200 mV). The second occurs only in mitochondria, is suggested to keep ΔΨ at low levels (100-150 mV) through the potential dependence of the ATP synthase and the allosteric ATP inhibition of cytochrome c oxidase at high ATP/ADP ratios, and is reversibly switched on by cAMP-dependent phosphorylation. Finally, the regulation of ΔΨ and the production of reactive oxygen species (ROS) in mitochondria at high ΔΨ values (150-200 mV) are discussed.     

Genetic diversity of nectar‐rewarding Platanthera chlorantha and nectarless Cephalanthera rubra

We examined the genetic diversity of two orchid species, the nectar‐rewarding Platanthera chlorantha and the nectarless Cephalanthera rubra, in north‐eastern Poland. We found lower differences in genetic diversity between the species than we expected. The level of genetic variation at species level was lower in C. rubra (P_POL = 14%, A = 1.14 and H_E = 0.060) than in P. chlorantha (P_POL = 25.5%, A = 1.35 and H_E = 0.078). In the majority of populations of both species a high proportion of unique genotypes was noted. The overall F_ST values for all populations were moderate and similar for both species (P. chlorantha: 0.251, P < 0.001; C. rubra: 0.267, P < 0.001). No relationship was found between genetic and geographical distances in either species (P > 0.05, Mantel test). We discuss the breeding systems, small population size and population subdivision as the most important factors affecting the genetic diversity of this species. We suggest that conservation programmes should be initiated to maintain or even increase the fitness and genetic variation of populations of both species. © 2013 The Linnean Society of London, Botanical Journal of the Linnean Society, 2013 , 171 , 751–763.     

Birth in winter can reduce the risk of lung cancer: A retrospective study of the birth season of patients with lung cancer in Beijing area,China

The season of birth is an important risk factor for several diseases. We explored the relationship between birth season and lung cancer. In this population-based retrospective study, we focused on patients with lung cancer who had registered at the Beijing Institute for Cancer Research from 2003 to 2012. In total, 33,025 patients were divided into five subgroups based on their histologic classification, and these five subgroups were compared with the general population (i.e., the permanent resident population of Beijing in 2013). A binary logistic regression method with sex and age as control factors was used to evaluate the relationship between birth season and lung cancer; P < 0.01 was statistically significant. Taking winter as a reference in our analysis of the relationship between season of birth and lung cancer, we found that people who were born in other seasons had a higher probability of developing lung cancer (spring: odds ratio [OR] = 1.06, 95% confidence interval [CI] = 1.03–1.09; summer: OR = 1.07, 95% CI = 1.04–1.10; autumn: OR = 1.06, 95% CI = 1.03–1.09) (P < 0.01). Among the five subgroups, persons with squamous cell carcinoma who were born in summer were more likely to develop lung cancer than those who were born in winter (OR = 1.09, 95% CI = 1.02–1.15, P = 0.006). The other subgroups showed no correlation with season of birth (P > 0.01). This study demonstrates that for people born in winter, the risks of developing lung cancer and squamous cell carcinoma are comparatively lower than those for people born in other seasons. Differences in immune function and the maternal nutrition status during pregnancy of people born in different seasons may explain this finding.     

Recovery of Populus tremuloides seedlings following severe drought causing total leaf mortality and extreme stem embolism

In contrast with other native Populus species in North America, Populus tremuloides (aspen) can successfully establish itself in drought‐prone areas, yet no comprehensive analysis has been performed on the ability of seedlings to withstand and recover from a severe drought resulting in complete leaf mortality. Here, we subjected 4‐month‐old aspen seedlings grown in two contrasting soil media to a progressive drought until total leaf mortality, followed by a rewatering cycle. Stomatal conductance (g_s), photosynthesis and transpiration followed a sigmoid decline with declining fraction of extractable soil water values. Cessation of leaf expansion occurred close to the end of the linear‐decrease phase, when g_s was reduced by 95%. Leaf mortality started after g_s reached the lowest values, which corresponded to a stem–xylem pressure potential (Ψ_xp) of ?2.0 MPa and a percent loss of stem hydraulic conductivity (PLC) of 50%. In plants with 50% leaf mortality, PLC values remained around 50%. Complete leaf mortality occurred at an average stem PLC of 90%, but all seedlings were able to resprout after 6–10 days of being rewatered. Plants decapitated at soil level before rewatering developed root suckers, while those left with a 4‐cm stump or with their stems intact resprouted exclusively from axillary buds. Resprouting was accompanied by recovery of stem hydraulic conductivity, with PLC values around 30%. The percentage of resprouted buds was negatively correlated with the stem %PLC. Thus, the recovery of stem hydraulic conductivity appears as an important factor in the resprouting capacity of aspen seedlings following a severe drought.     

PAX5 gene as a novel methylation marker that predicts both clinical outcome and cisplatin sensitivity in esophageal squamous cell carcinoma

Therapeutic strategies for esophageal cancer largely depend on histopathological assessment. To select appropriate treatments of individual patients, we examined the background molecular characteristics of tumor malignancy and sensitivity to multidisciplinary therapy. Seventy-eight surgically-resected esophageal squamous cell carcinoma (ESCC) cases during 2001–2013 were examined. PAX5, a novel gene methylation marker in ESCC, was evaluated in the specimens, as methylation of this gene was identified as an extremely tumor-specific event in squamous cell carcinogenesis of head and neck. PAX5 methylation status was evaluated by quantitative MSP (QMSP) assays. Mean QMSP value was 15.7 (0–136.3) in ESCCs and 0.3 (0–8.6) in adjacent normal tissues (P < 0.001). The 78 cases were divided into high QMSP value (high QMSP, n = 26) and low QMSP value (low QMSP, n = 52). High QMSP cases were significantly associated with downregulated PAX5 expression (P = 0.040), and showed significantly poor recurrence-free survival [Hazard Ratio (HR) = 2.84; P = 0.005; 95% Confidence Interval (CI): 1.39–5.81] and overall survival (HR = 3.23; P = 0.002; 95%CI: 1.52–7.01) in multivariable analyses with histopathological factors. PAX5-knockdown cells exhibited significantly increased cell proliferation and cisplatin resistance. PAX5 gene methylation can predict poor survival outcomes and cisplatin sensitivity in ESCCs and could be a useful diagnostic tool for cancer therapy selection.     

A synergistic role for two predicted inner membrane proteins of Escherichia coli in cell envelope integrity

The bacterial cytoplasmic membrane is a principal site of protein translocation, lipid and peptidoglycan biogenesis, signal transduction, transporters and energy generating components of the respiratory chain. Although 25–30% of bacterial proteomes consist of membrane proteins, a comprehensive understanding of their influence on fundamental cellular processes is incomplete. Here, we show that YciB and DcrB, two small cytoplasmic membrane proteins of previously unknown functions, play an essential synergistic role in maintaining cell envelope integrity of Escherichia coli. Lack of both YciB and DcrB results in pleiotropic cell defects including increased levels of lipopolysaccharide, membrane vesiculation, dynamic shrinking and extension of the cytoplasmic membrane accompanied by lysis and cell death. The stalling of an abundant outer membrane lipoprotein, Lpp, at the periplasmic face of the inner membrane leads to lethal inner membrane–peptidoglycan linkages. Additionally, the periplasmic chaperone Skp contributes to yciB dcrB mutant cell death by possibly mistargeting stalled porins into the inner membrane. Consistent with the idea of a compromised envelope in the yciB dcrB mutant, multiple envelope stress response systems are induced, with Cpx signal transduction being required for growth. Taken together, our results suggest a fundamental role for YciB and DcrB in cell envelope biogenesis.     

Limits to xylem refilling under negative pressure in Laurus nobilis and Acer negundo

The ability of juvenile Laurus nobilis and Acer negundo plants to refill embolized xylem vessels was tested under conditions of soil drought when xylem sap pressure was substantially negative, thus violating the expected condition that pressure must rise to near atmospheric for refilling. Intact potted plants were dried to a stem water potential (Σ_W) corresponding with approximately 80% loss of hydraulic conductivity (PLC) in shoots. Then plants were re‐watered and kept at a less negative target Ψ_W for 1–48 h. The Ψ_W was measured continuously with stem psychrometers. Rewatered L. nobilis held at the target Ψ_W for 1 h showed no evidence for refilling unless Ψ_W was within a few tenths of a MPa of zero. In contrast, re‐watered L. nobilis held for 24 and 48 h at water potentials well below zero showed a significant reduction in PLC. The recovery was highly variable, being complete in some stem segments, and scarcely evident in others. Embolism repair was accompanied by a significant but moderate decrease in the osmotic potential (Ψ) of the bulk xylem sap (Ψ = ?67 kPa in recovering plants versus ?31 kPa in controls). In contrast, embolized and re‐watered A. negundo plants held for 24 h at target Ψ_W of ?0·9 and ?0·3 MPa showed no embolism reversal. The mechanism allowing L. nobilis plants to refill under negative pressure is unknown, but does not appear to operate in A. negundo, and is slower to act for drought‐induced embolism than when embolism was artificially induced by air injection as previously shown for L. nobilis.     

Single‐nucleotide Polymorphism of CD36 Locus and Obesity in European Adolescents

CD36 is a membrane receptor with a wide variety of functions, including the regulation of energy metabolism, fat storage, and adipocyte differentiation. To assess the relationship between CD36 gene single‐nucleotide polymorphisms (SNPs) and obesity in adolescents, we evaluated the relationship between CD36 SNPs and the risk of obesity in a case–control study composed of 307 obese (age = 15.0 ± 1.1 years) and 339 normal‐weight adolescents (age = 14.6 ± 1.1 years). To validate the results, we assessed the relation between the same SNPs and percentage of body fat (BF%) and BMI in 1,151 European adolescents (age = 14.8 ± 1.4 years). SNPs with a minor allele frequency >0.10 were selected to tag CD36. Genotyping was performed on an Illumina system. Four SNPs (rs3211867, rs3211883, rs3211908, and rs1527483) were associated with increased risk of obesity in the case–control study (odds ratio (OR) (95% confidence interval)): 1.96 (1.26–3.04], P = 0.003; 1.73 (1.16–2.59), P = 0.007; 2.42 (1.47–4.01), P = 0.0005 and 1.95 (1.25–3.05), P = 0.003, respectively). The same four SNPs were associated with higher BMI (P < 0.05) and BF% (P < 0.04) in the validation study. Further analyses identified a haplotype (frequency: 0.05) carrying the minor allele of these SNPs as being associated with obesity (OR: 2.28; P = 0.0008) in the case–control study and with excess adiposity (i.e., higher BF% (P = 0.03) and BMI (P = 0.04)) in the validation study. Our data suggest that genetic variability at the CD36 gene locus could be associated with body weight variability in European adolescents but these findings require replication.     

Germination of Prosopis juliflora (Sw.) D.C. seeds at different osmotic potentials and temperatures

The effects of osmolytes, osmotic potential (Ψ_s), temperature, and their interactions on the germinability, germination rate, and other germination parameters of the invasive shrub Prosopis juliflora, which grows in the semiarid environmental conditions of the Caatinga in northeast Brazil, were evaluated. To study the effects of polyethylene glycol (PEG) and NaCl stress and temperature on germination, two separate experiments were carried out at the Plant Ecophysiology Laboratory of the Federal University of Pernambuco in 2011. The overall germinability decreased significantly with increases in both PEG (one‐way anova , F_4,75 = 111.21, P ≤ 0.001) and NaCl (one‐way anova , F_4,75 = 12.82, P ≤ 0.001); however, the effects were more accentuated with PEG than NaCl. The PEG‐treated seeds maintained their germinability, even when they were subjected to a Ψ_s = ?1 MPa after being rinsed and allowed to germinate on deionized water. In contrast, NaCl‐treated seeds usually lost their ability to germinate; this fact was possibly linked to the accumulation of Na⁺ and Cl^? in the cells, which may have contributed to a loss of membrane function that led to the death of the embryos. Although numerous studies describing seed germination in the presence of osmolytes have been conducted, studies that show the interactions between osmolytes, osmotic potentials, and temperature are scarce. The present study is the first to describe these interactions for P. juliflora seeds.     

Membrane permeabilities determining resting,action and mechanoreceptor potentials inStentor coeruleus

Summary In response to mechanical stimuli the protozoan,Stentor coeruleus, contracts in an all-or-none fashion and simultaneously reverses the direction of its ciliary beat. These behaviors have previously been shown to be correlated with the presence of a mechanoreceptor potential and all-or-none action potential (Wood 1970, 1973a). In the studies reported below the ionic bases of the resting, receptor and action potentials ofStentor were determined by use of intracellular microelectrodes penetrating animals chilled to 8.5–10 °C. The resting potential is most dependent on the extracellular concentration of KCl but some dependence on CaCl₂ concentration was also observed. If allowance is made for the large increases in membrane conductance observed in solutions containing 2–8 mM KCl it is found that the resting potential data are well described by a modified form of the Goldman equation whereP _Ca/P _K = 0.068 andP _Cl/P _K = 0.072. The intracellular ionic activities (K _i ⁺ = 13.1 mM, Cl _i ^– = 9.9 mM, Ca _i ⁺ = 0 mM) which provide the best fit of this equation to the resting potential data are in close agreement with the intracellular concentration values measured by flame microspectrophotometry (K_i=12.4 mM, Cl_i = 9.4 mM) except in the case of Ca_i where most of the intracellular concentration is presumed to be bound. 65 to 75 mV action potentials are produced by suprathreshold depolarizations but contractions were not generally seen in these chilled animals, only ciliary reversals. The action potential peak varies with CaCl₂ concentration with a slope of 12.6 mV/10 fold change but varies only slightly with KCl or Cl^– concentration. These peak potentials are well described by assuming that theP _Ca/P _k = 7.9 andP _Cl/P _K=1.0 at the time of the action potential peak. Depolarizing receptor potentials and brief inward receptor currents were observed for all forms of punctate and gross bodily mechanical stimulation employed. No evidence was found for any form of hyperpolarizing mechanoreceptor potentials as observed in some other ciliates. The reversal potential of the mechanoreceptor current varied with CaCl₂ concentration in a manner similar to that of the action potential peak. As in the case of the action potential both theP _Ca/P _k andp _cl/p _k ratios appear to increase as a result of mechanical stimulation to 9.3–15 and 1.2–1.95 respectively. Mechanoreceptor currents are voltage dependent being increased when the membrane is depolarized above resting potential and decreased when the membrane is hyperpolarized. In general the electrophysiological characteristics ofStentor appear similar to those ofParamecium andStylonychia, but its resting membrane appears more selectively permeable to K⁺, it produces only depolarizing receptor potentials when mechanically stimulated and the initial action potential elicited by depolarizing current pulses can be all-or-none even in culture medium.     

Lateral gradients significantly enhance static magnetic field‐induced inhibition of pain responses in mice—a double blind experimental study

Recent research demonstrated that exposure of mice to both inhomogeneous (3–477 mT) and homogeneous (145 mT) static magnetic fields (SMF) generated an analgesic effect toward visceral pain elicited by the intraperitoneal injection of 0.6% acetic acid. In the present work, we investigated behavioral responses such as writhing, entry avoidance, and site preference with the help of a specially designed cage that partially protruded into either the homogeneous (ho) or inhomogeneous (inh) SMF. Aversive effects, cognitive recognition of analgesia, and social behavior governed mice in their free locomotion between SMF and sham sides. The inhibition of pain response (I) for the 0–5, 6–20, and 21–30 min periods following the challenge was calculated by the formula I = 100 (1 ? x/y) in %, where x and y represent the number of writhings in the SMF and sham sides, respectively. In accordance with previous measurements, an analgesic effect was induced in exposed mice (I_ho = 64%, P < 0.0002 and I_inh = 62%, P < 0.002). No significant difference was found in the site preference (SMF_{ho, inh} vs. sham) indicating that SMF is neither aversive nor favorable. Comparison of writhings observed in the sham versus SMF side of the cage revealed that SMF exposure resulted in significantly fewer writhings than sham (I_ho = 64%, P < 0.004 and I_inh = 81%, P < 0.03). Deeper statistical analysis clarified that the lateral SMF gradient between SMF and sham sides could be responsible for most of the analgesic effect (I_ho = 91%, P < 0.02 and I_inh = 54%, P < 0.02). Bioelectromagnetics 34:385–396, 2013. © 2012 Wiley Periodicals, Inc.     

Zinc content of normal human serum and its correlation with some hematic parameters

Numerous studies have demonstrated that several diseases and stress conditions are associated with changes in the levels of zinc in the blood plasma and cellular elements. In this research the association between serum zinc concentrations and other hematic parameters of diagnostic interest has been evaluated. Quantitative determinations of zinc, total plasmatic proteins, albumin, hemoglobin and calculation of mean corpuscular volume were performed on blood samples from 58 males aged 20–61 years. Concentrations measured in our sample are comparable with reference values. Statistically significant correlation coefficients were found between age and albumin (r = - 0.562, P < 0.001), serum zinc and albumin (r = 0.328, P < 0.05), serum zinc and hemoglobin (r = 0.291, P < 0.05), and total plasmatic proteins and albumin (r = 0.463, P < 0.001). These correlation coefficients were significant even after adjustment for age effect. The determination of serum zinc concentration may be useful in the assessment of clinical scenarios. Particularly, it may provide additional information for the diagnosis of specific pathologies, such as hepatic malfunctions. It could also be useful in the identification of different stages of anemia.     

Dynamics of leaf water relations components in co‐occurring iso‐ and anisohydric conifer species

Because iso‐ and anisohydric species differ in stomatal regulation of the rate and magnitude of fluctuations in shoot water potential, they may be expected to show differences in the plasticity of their shoot water relations components, but explicit comparisons of this nature have rarely been made. We subjected excised shoots of co‐occurring anisohydric Juniperus monosperma and isohydric Pinus edulis to pressure‐volume analysis with and without prior artificial rehydration. In J. monosperma, the shoot water potential at turgor loss (Ψ_TLP) ranged from ?3.4 MPa in artificially rehydrated shoots to ?6.6 MPa in shoots with an initial Ψ of ?5.5 MPa, whereas in P. edulis mean Ψ_TLP remained at ～ ?3.0 MPa over a range of initial Ψ from ?0.1 to ?2.3 MPa. The shoot osmotic potential at full turgor and the bulk modulus of elasticity also declined sharply with shoot Ψ in J. monosperma, but not in P. edulis. The contrasting behaviour of J. monosperma and P. edulis reflects differences in their capacity for homeostatic regulation of turgor that may be representative of aniso‐ and isohydric species in general, and may also be associated with the greater capacity of J. monosperma to withstand severe drought.     

Coagulation inhibitors and glycaemic control in non-insulin-dependent diabetes mellitus

The relationship between long-term glycaemic control and the activity of coagulation inhibitors was investigated in 60 non-insulin-dependent diabetes mellitus (NIDDM) patients not on insulin therapy. Overall, the activities of antithrombin III (AT III) (median 96%, range 65–133%), protein C (127%, 24–190%) and protein S (130%, 54–163%) were not reduced. Patients in poor long-term glycaemic control as verified by increased glycated haemoglobin (HbA1c) demonstrated significantly decreased median AT III activity in comparison with patients in good glycaemic control (92% vs 101%,P=0.016). However, individual values for AT III activity were not below the critical limit of 60%. An inverse correlation between AT III activity and long-term glycaemic control (HbA1c) was calculated (r=–0.378,P=0.0029). As AT III concentrations were found to be normal, we propose that non-enzymatic glycation leads to reduced activity of AT III without affecting its concentration.     

Does the turgor loss point characterize drought response in dryland plants?

The water potential at turgor loss point (Ψ_tlp) has been suggested as a key functional trait for determining plant drought tolerance, because of its close relationship with stomatal closure. Ψ_tlp may indicate drought tolerance as plants, which maintain gas exchange at lower midday water potentials as soil water availability declines also have lower Ψ_tlp. We evaluated 17 species from seasonally dry habitats, representing a range of life‐forms, under well‐watered and drought conditions, to determine how Ψ_tlp relates to stomatal sensitivity (pre‐dawn water potential at stomatal closure: Ψg_s0) and drought strategy (degree of isohydry or anisohydry; ΔΨ_MD between well‐watered conditions and stomatal closure). Although Ψg_s0 was related to Ψ_tlp, Ψg_s0 was better related to drought strategy (ΔΨ_MD). Drought avoiders (isohydric) closed stomata at water potentials higher than their Ψ_tlp; whereas, drought tolerant (anisohydric) species maintained stomatal conductance at lower water potentials than their Ψ_tlp and were more dehydration tolerant. There was no significant relationship between Ψ_tlp and ΔΨ_MD. While Ψ_tlp has been related to biome water availability, we found that Ψ_tlp did not relate strongly to stomatal closure or drought strategy, for either drought avoiders or tolerators. We therefore suggest caution in using Ψ_tlp to predict vulnerability to drought.     

Maternal Behavior Indicates Survival and Cause-Specific Mortality of Moose Calves

Continuing research on cause-specific mortality and annual survival of moose (Alces alces) calves in northeastern Minnesota, USA, is important to understanding the long-term trajectory of the population. In 2013 and 2014, we observed global positioning system (GPS)-collared, female moose exhibit a specific behavior (i.e., mortality movement) associated with the death of their GPS-collared neonate. The females made a rapid, long-distance movement (flee), followed by a return to the calf mortality site. We used characteristics of this movement in 2013–2014 (n = 46) to develop models for assessing calf survival, and then evaluated these models using female movement rates (n = 49) in 2015−2016. Using this behavior as an indicator of calf mortality in 2016, we conducted field investigations, leading to evidence of 15 mortalities at a mean age of 30.6 ± 15.5 (SE) days (range = 3–243 days). We launched 21 investigations in response to a mortality movement and they resulted in confirmation of 11 of the 15 calf mortalities. Specific causes of mortality included 9 wolf (Canis lupus)-kills, 3 black bear (Ursus americanus)-kills, 1 unknown predator-kill, and 2 deaths following vehicle collisions. The mean distance females fled after a mortality was 1,873 ± 412 m (range = 126–5,805 m, n = 14). Females that made return visits returned a mean 2.8 ± 0.5 times (range = 1–5, n = 8) to within a mean 106 ± 22 m (range = 34–230 m, n = 8) of the mortality site. Calf survival to 30 days of age was 67 ± 8% (95% CI = 53–84%, n = 36) but declined to 53 ± 8% (95% CI = 39–72%, n = 36) by 3 months of age. We developed 2 population-level movement models to improve the efficacy of using the mortality movement to identify and locate calf mortalities in real time via field investigations. The first approach, a temporal-based model, used a 3-day average movement velocity threshold (118 m/hr) for all females to indicate calf mortality and accurately predicted survival status in 51% (n = 105) of the cases. The second approach, an age-specific model using different thresholds (28–135 m/hr) for females relative to calf age, was 80% (n = 231) accurate. Using movement behavior of females to assess calf mortality yielded important insights into mechanisms influencing the population decline that will inform future management decisions. © 2019 The Wildlife Society     

Apoplasmic water fractions and osmotic potentials at full turgidity of some Bryidae

The fractions of apoplasmic water of six moss species were estimated by comparison of the osmotic potentials of fully turgid living tissues (Ψ _π(s)) and of killed shoots (Ψ _π(k)). The values of Ψ _π(s) were determined by pressure-volume analysis using thermocouple hygrometry, whereas those of Ψ _π(k) were obtained by cryoscopy using extracts produced from dried materials and taking into consideration the original saturation water content of the shoots. Most of the tissues had Ψ _π(s) values around-1 to -1.5 MPa and non-osmotic water fractions of roughly 20% of the total water content at full turgor. Quantitative analysis of a number of osmotically active cell constituents showed that about one-third of the osmotic potential resulted from the accumulation of sugars. The total free amino acids accounted for about 15–20% of the osmolality of the cell solution. Malic- and citricacid contents varied much more with species but, in general, both carboxylic acids together contributed nearly 10–20% to the osmotic potential. The contents of inorganic anions such as free chloride and phosphate in mosses were low.     

Conformational States of the Water-Soluble Fragment of Cytochrome b5. I. pH-Induced Denaturation

Cytochrome b ₅ is a membrane protein that comprises two fragments: one is water-soluble and heme-containing, and the other is hydrophobic and membrane-embedded. The function of electron transfer is performed by the former whose crystal structure is known; however, its conformational states when in the membrane field and interacting with other proteins are still to be studied. Previously, we proposed water–alcohol mixtures for modeling the effect of membrane surface on proteins, and used this approach to study the conformational behavior of positively charged cytochrome c as well as relatively neutral retinol-binding protein also functioning in the field of a negatively charged membrane. The current study describes the conformational behavior of the negatively charged water-soluble fragment of cytochrome b ₅ as dependent on pH. Decreasing pH was shown to transform the fragment state from native to intermediate, similar to the molten globule reported earlier for other proteins in aqueous solutions: at pH 3.0, the fragment preserved a pronounced secondary structure and compactness but lost its rigid tertiary structure. A possible role of this intermediate state in cytochrome b ₅ functioning is discussed.