Evolutionary Role of Iron in Metabolism of Prokaryotes and in Biogeochemical Processes

The paper reviews data summarizing points of view about the conceptual role of iron in the appearance and evolutionary formation of the Earth and its biosphere. Participation of iron and its compounds in the appearance and development of processes of anaero- and aerobiosis as fundamental blocks of metabolism is presented as a hierarchical scheme. Magnetically arrayed iron compounds, in which the element is both in the Fe(II) and in the Fe(III) state, are considered a connecting link between the hierarchical levels. It is shown that the energy transformation Fe(II) Fe(III) is an oxidation–reduction energy core of the most important metabolic iron complexes and of processes of biogenesis both at the cellular level and in biogeosystems.     

Mechanism of neurofibrillary degeneration in Alzheimer's disease

Neurofibrillary degeneration associated with the formation of intraneuronal neurofibrillary tangles of paired helical filaments (PHF) and 2.1 nm τ filaments is one of the most characteristic brain lesions of Alzheimer's disease. The major polypeptides of PHF are the microtubule associated protein, τ. τ, in PHF is present in abnormally phosphorylated forms. In addition to the PHF, the abnormal τ is present in soluble non-PHF form in the alzheimer's disease brain. The level of τ in Alzheimer's disease neocortex is severalfold higher than in aged control brain, and this increase is in the form of the abnormally phosphorylated protein. The abnormally phosphorylated τ does not promote the assembly of tubulin into microtubules in vitro, and it inhibits the normal τ-stimulated microtubule assembly. After in vitro dephosphorylation both PHF and non-PHF abnormal τ stimulate the assembly of tubulin into microtubules. The activities of phosphoseryl/phosphothreonyl protein phosphatase 2A and nonreceptor phosphotyrosyl phosphatase(s) are decreased in AD brain. It is suggested that

1. A defect(s) in the protein phosphorylation/dephosphorylation system is one of the early events in the neurofibrillary pathology in AD;
2. A decrease in protein phosphatase, activities, at least in part, allows the hyperphosphorylation of τ; and
3. Abnormal phosphorylation and polymerization of τ into PHF most probably lead to a breakdown of the microtubule system and consequently to neuronal degeneration.

     

Changes in the level of oxygen tension in different brain structures of rats in positive and negative emotional states

Changes of oxygen tension (pO2) level in various brain structures were studied in rats in positive and negative emotional states. It is established that pO2 level changes depend on the character of behavioural reactions: "active" type of behaviour (emotionally positive orienting-investigatory, actively defensive) is accompanied by pO2 level increase, and "passive" type of behaviour ("freezing" reaction)--by a decrease of pO2 level. Changes of oxidative brain metabolism observed at "active" and "passive" types of behaviour indicate, respectively, adaptive and nonadaptive character of these behavioural reactions.     

Functional Characterization of a Silicon Transporter Gene Implicated in Silicon Distribution in Barley

Silicon (Si) is a beneficial element for plant growth. In barley (Hordeum vulgare), Si uptake by the roots is mainly mediated by a Si channel, Low Silicon1 (HvLsi1), and an efflux transporter, HvLsi2. However, transporters involved in the distribution of Si in the shoots have not been identified. Here, we report the functional characterization of a homolog of HvLsi1, HvLsi6. HvLsi6 showed permeability for Si and localized to the plasma membrane. At the vegetative growth stage, HvLsi6 was expressed in both the roots and shoots. The expression level was unaffected by Si supply. In the roots, HvLsi6 was localized in epidermis and cortex cells of the tips, while in the leaf blades and sheaths, HvLsi6 was only localized at parenchyma cells of vascular bundles. At the reproductive growth stage, high expression of HvLsi6 was also found in the nodes. HvLsi6 in node I was polarly located at the transfer cells surrounding the enlarged vascular bundles toward the numerous xylem vessels. These results suggest that HvLsi6 is involved in Si uptake in the root tips, xylem unloading of Si in leaf blade and sheath, and intervascular transfer of Si in the nodes. Furthermore, HvLsi2 was found to be localized at the parenchyma cell layer adjacent to the transfer cells with opposite polarity of HvLsi6, suggesting that the coupling of HvLsi6 and HvLsi2 is involved in the intervascular transfer of Si at the nodes. Si translocated via the enlarged vascular bundles is unloaded to the transfer cells by HvLsi6, followed by HvLsi2 to reload Si to the diffuse vascular bundles, which are connected to the upper part of the plant, especially the panicles, the ultimate Si sink.Silicon (Si) is a beneficial element for plant growth. It enhances the resistance of plants to various biotic and abiotic stresses (Epstein, 1999; Ma and Takahashi, 2002; Ma and Yamaji, 2006). For example, Si reduces the epidemics of both leaf and panicle blast in rice (Oryza sativa; Datnoff and Rodrigues, 2005) and decreases the incidence of powdery mildew in cucumber (Cucumis sativus), barley (Hordeum vulgare), and wheat (Triticum aestivum; Fauteux et al., 2005). Si also suppresses insect pests such as stem borer (Chilo suppressalis), brown planthopper (Nilaparvata lugens), and rice green leafhopper (Nephotettix cincticeps; Savant et al., 1997). Resistance to the damage by wild rabbit in wheat is also enhanced by an increased amount of Si in leaf tissue (Cotterill et al., 2007). Si is also able to alleviate lodging, drought, and low- and high-temperature stresses (Ma, 2004). The beneficial effects of Si under phosphate deficiency, phosphate excess, and manganese and salt toxicity stresses have been observed in many plants (Ma and Takahashi, 2002). Usually, the more Si that accumulates in the shoots, the greater its effect in enhancing the plant’s response. This is because most effects of Si are expressed through the formation of silica gel, which is deposited on leaves, stems, and other organs of plants (Ma and Yamaji, 2006). Therefore, for the plant to benefit from Si, a high accumulation is required. However, Si accumulation greatly varies with plant species, and this difference has been attributed to the ability of plants to take up Si.Transporters responsible for Si uptake by roots have been identified in several plant species, including barley, maize (Zea mays), pumpkin (Cucurbita moschata), rice, wheat (Ma et al., 2011), and most recently in horsetail (Equisetum arvense; EaNIP3s [for Nod26-like major intrinsic protein3]; Grégoire et al., 2012). Two different types of transporter, Si-permeable channel and efflux transporter, are involved in the Si-uptake process. Low Silicon1 (Lsi1) belongs to a NIP subfamily of aquaporin-like proteins and functions as a Si-permeable channel. Lsi1 in rice is localized in the distal side of root exodermis and endodermis (Ma et al., 2006), but Lsi1 in barley, maize, and pumpkin is localized in the epidermis and cortex (Chiba et al., 2009; Mitani et al., 2009b, 2011). On the other hand, Lsi2 functions as an efflux Si transporter and belongs to a putative anion transporter family without any similarity to Lsi1. Lsi2 in rice is also localized at the root exodermis and endodermis as Lsi1, but it is polarly localized at the proximal side (Ma et al., 2007). By contrast, Lsi2 in barley and maize is localized only to the endodermis of roots. Furthermore, these transporters do not show polar localization in barley and maize (Mitani et al., 2009a). Therefore, Si uptake mediated by Lsi1 and Lsi2 shows different pathways between rice and other plant species (Ma et al., 2011).Following uptake by the roots through Lsi1 and Lsi2, Si is translocated to the aboveground part and distributed in different tissues. Lsi6, a homolog of Lsi1, is involved in xylem unloading of Si in rice (Yamaji et al., 2008). Lsi6 is localized on the adaxial side of the xylem parenchyma cells in the leaf sheaths and leaf blades. Knockout of Lsi6 resulted in altered distribution of Si in the leaf cells. Furthermore, at the reproductive growth stage of rice, Lsi6 is also highly expressed at the nodes (Yamaji and Ma, 2009). At node I below the panicle, Lsi6 is mainly localized at the xylem transfer cells with polarity facing toward the xylem vessel (Yamaji and Ma, 2009). Knockout of Lsi6 decreased Si accumulation in the panicle but increased Si accumulation in the flag leaf. These findings indicate that Lsi6 is also required for the intervascular transfer of Si in rice, transferring Si from the enlarged vascular bundles coming from the roots to the diffuse vascular bundles connected to the panicle.Barley is a Si-accumulating species, although the accumulation extent is lower than that of rice. Transporters responsible for Si uptake in barley roots have been identified (Chiba et al., 2009; Mitani et al., 2009a); however, transporters for Si distribution in aboveground plant tissues are unknown. In this study, we functionally characterized a rice Lsi6 homolog gene in barley, HvLsi6, in terms of transport activity and expression pattern, as well as cellular and subcellular localizations. We found that HvLsi6 is probably involved in Si uptake in the root tip, xylem unloading in the leaf, and intervascular transfer of Si at the nodes in barley. We further found that HvLsi2 was also expressed in the nodes and involved in the intervascular transfer by coupling with HvLsi6.     

Model of mechanical alternans in the mammalian myocardium

A model is proposed to elucidate the cause and mechanism of mechanical alternans in cardiac muscle in terms of discrete calcium movements. Mechanical alternans, the cause of which lies within the borders of excitation-contraction-coupling (ECC), is analyzed. In this case, the "input" of the ECC system (the action potentials and intervals) is constant while the "output" (contractile force) oscillates between two constant values, indicating that the system has a "memory" with two "internal states". It is proposed that these two "states" are associated with a part of the sarcoplasmic reticulum ("releasable terminal") containing the readily releasable calcium. A mechanism of "calcium-concentration-dependent threshold" is suggested to govern the "release function", i.e. the release of calcium from the "releasable terminal" to the myofilaments. The "release function" is analyzed in both the linear and the non-linear cases and its implication on the initiation of sustained and transient mechanical alternans are described. The dependence of mechanical alternans on a disturbance is also explained. The model response resembles the experimental observations of mechanical alternans in mammalian myocardium in the following manners: abrupt transition from low to high heart rates, slow progressive acceleration of rate, variations in persistence at subthreshold rates, effect of premature and delayed beat following the small and large beats, restitution curves, and transient mechanical alternans initiated by a delayed beat.     

Evaluation of Buprenorphine in a Postoperative Pain Model in Rats

We evaluated the commonly prescribed analgesic buprenorphine in a postoperative pain model in rats, assessing acute postoperative pain relief, rebound hyperalgesia, and the long-term effects of postoperative opioid treatment on subsequent opioid exposure. Rats received surgery (paw incision under isoflurane anesthesia), sham surgery (anesthesia only), or neither and were treated postoperatively with 1 of several doses of subcutaneous buprenorphine. Pain sensitivity to noxious and nonnoxious mechanical stimuli at the site of injury (primary pain) was assessed at 1, 4, 24, and 72 h after surgery. Pain sensitivity at a site distal to the injury (secondary pain) was assessed at 24 and 72 h after surgery. Rats were tested for their sensitivity to the analgesic and locomotor effects of morphine 9 to 10 d after surgery. Buprenorphine at 0.05 mg/kg SC was determined to be the most effective; this dose induced isoalgesia during the acute postoperative period and the longest period of pain relief, and it did not induce long-term changes in opioid sensitivity in 2 functional measures of the opioid system. A lower dose of buprenorphine (0.01 mg/kg SC) did not meet the criterion for isoalgesia, and a higher dose (0.1 mg/kg SC) was less effective in pain relief at later recovery periods and induced a long-lasting opioid tolerance, indicating greater neural adaptations. These results support the use of 0.05 mg/kg SC buprenorphine as the upper dose limit for effective treatment of postoperative pain in rats and suggest that higher doses produce long-term effects on opioid sensitivity.Relief of postoperative pain is mandated in the Guide for the Care and Use of Animals¹⁸ and the Public Health Service Policy¹⁷ and is a major objective of laboratory animal medicine. Buprenorphine is one of the most commonly used opioid analgesics for postoperative pain in laboratory animals, mainly because of its long duration of action.¹⁰ The typical recommended dose range of buprenorphine in rats is 0.02 to 0.05 mg/kg SC.¹⁰ The upper end of this range, although effective at relieving acute postoperative pain in rats, is associated with side effects such as enhanced postoperative pain after the drug has worn off (rebound hyperalgesia),²³ respiratory depression,²¹ nausea or gastrointestinal distress and pica,²⁵ and neural adaptations (for example, sensitization) that may lead to long-term changes in neural function in the central nervous system and consequent changes in behavior.¹⁴ Central sensitization is a well-studied neural adaptation expressed in the brain and spinal cord and induced by nociceptive stimulation (that is, pain-induced by surgical manipulation) that manifests as hyperalgesia (decreased pain threshold to noxious stimuli) and allodynia (appearance of pain-like responses to nonnoxious tactile stimuli) during the recovery period.^16,29 Central sensitization contributes to persistent pain during the postoperative recovery period (that is, maintenance of increased pain sensitivity during tissue recovery) and chronic pain in some pathologic conditions (that is, persistent pain sensitivity after full tissue recovery). Central sensitization also accounts for the spread of hyperalgesia and allodynia to noninjured areas of the body distal to the injury.³¹ This phenomenon is referred to as ‘secondary pain’ (secondary hyperalgesia and allodynia), because it is not directly associated with the primary injury site.Opioid analgesics inhibit pain by acting on the nervous system to block transduction of pain signals traveling in sensory neurons toward the central nervous system and by facilitating activity of the descending pain inhibition neural pathway.¹⁶ Opioid analgesics also induce neural adaptations in the nervous system, phenomena that underlie the pronounced changes in behavior associated with addiction to narcotics.² Notably, opioid analgesics have been shown to enhance central sensitization initiated by pain transmission.^6,8,14,20 This property means that opiate analgesics facilitate both the inhibition of pain and central sensitization that leads to the enhancement of pain. Because central sensitization is a neural adaptation, the interaction of opiates on this pain mechanism outlasts the presence of the drug; in contrast, opiate effects on pain inhibition are limited to the presence of the drug. This arrangement is thought to account for rebound pain, that is, increased pain sensitivity after the opiate analgesic has worn off. Opiate side effects can compromise the success of recovery by increasing the level of distress experienced during recovery (for example, inducing nausea) and possibly increasing the duration of distress during recovery (for example, allowing for rebound pain). Moreover, and of importance specifically to laboratory animal medicine, the general neural adaptations induced by even a single dose of an opiate analgesic²⁶ may induce changes in the nervous system that alter and therefore compromise the validity of the animal model under study (for example, opioid mechanisms involved in behavioral control).We previously evaluated the feasibility of oral administration of buprenorphine.^15,25 As a basis for comparison, we used the ‘gold-standard’ postoperative buprenorphine dose of 0.05 mg/kg SC. The results of those studies showed that oral administration of buprenorphine was not feasible because the dose necessary to produce analgesia comparable to the standard dose of 0.05 mg/kg SC was 10 times the oral dose recommended in the literature and because the resulting concentration of oral buprenorphine was too bitter for rats to ingest voluntarily in a volume of flavored foodstuff that they could eat in a single meal.^15,25 We also observed that both subcutaneous and oral buprenorphine caused conditioned aversion to flavors,²⁵ suggestive of gastrointestinal distress⁵, with a greater effect for the oral route. Our conclusions and the associated clinical recommendation were limited by our presumption that buprenorphine at 0.05 mg/kg SC was the ideal postsurgical dose.An assessment of the literature that established this dose identified 2 problems. First, little or no research had directly assessed the effect of buprenorphine on pain sensitivity in animals in the hyperalgesic state that characterized the postoperative period,²³ and to our knowledge, no study has directly assessed the dose–response function of postsurgical buprenorphine on hyperalgesia. We hypothesized that endogenous opioids activated during the postoperative period²⁴ might act synergistically with buprenorphine to allow adequate relief of postoperative pain with a lower dose of buprenorphine than is necessary in an algesiometric test, thereby making predictions and extrapolations from algesiometric tests inaccurate. Second, we found that little consideration had been given to the consequences of other physiologic effects of buprenorphine on the recovery process (for example, gastrointestinal distress⁵, rebound hyperalgesia, and allodynia). As stated earlier, recent research on central sensitization has determined that although opioid analgesics inhibit pain sensation acutely, they also enhance neural adaptations that account for rebound pain and other long-term chronic pain conditions.^16,28,29,31 We hypothesized secondarily that a lower dose of buprenorphine, if effective acutely, would result in reduced side effects and be less likely to initiate or enhance neural adaptations, such as rebound hyperalgesia and allodynia.The current study had 2 goals. The first was to establish the minimum dose of buprenorphine needed to relieve acute postoperative pain effectively in rats. As a starting point, we defined effective relief of acute pain as the induction of isoalgesia during the postoperative period; isoalgesia is the normal level of pain sensation, in contrast to analgesia (absence of pain sensation) or hypoalgesia (lower-than-normal pain sensation). The second goal was to evaluate the effect of postoperative buprenorphine on factors that slow recovery (that is, rebound hyperalgesia and allodynia) or create long-term changes (that is, sensitization or tolerance to opiates). We tested our hypothesis by using various doses of buprenorphine in a rat model of incisional pain.^3,4,31 This model was selected because it induces cutaneous and muscular pain common to most surgery and generates mild to moderate persistent pain so that both the acute inhibitory effects of the buprenorphine (that is, pain relief) and the lasting effects of buprenorphine (that is, rebound hyperalgesia) could be studied.     

Promises and Challenges of Eco-Physiological Genomics in the Field: Tests of Drought Responses in Switchgrass

Identifying the physiological and genetic basis of stress tolerance in plants has proven to be critical to understanding adaptation in both agricultural and natural systems. However, many discoveries were initially made in the controlled conditions of greenhouses or laboratories, not in the field. To test the comparability of drought responses across field and greenhouse environments, we undertook three independent experiments using the switchgrass reference genotype Alamo AP13. We analyzed physiological and gene expression variation across four locations, two sampling times, and three years. Relatively similar physiological responses and expression coefficients of variation across experiments masked highly dissimilar gene expression responses to drought. Critically, a drought experiment utilizing small pots in the greenhouse elicited nearly identical physiological changes as an experiment conducted in the field, but an order of magnitude more differentially expressed genes. However, we were able to define a suite of several hundred genes that were differentially expressed across all experiments. This list was strongly enriched in photosynthesis, water status, and reactive oxygen species responsive genes. The strong across-experiment correlations between physiological plasticity—but not differential gene expression—highlight the complex and diverse genetic mechanisms that can produce phenotypically similar responses to various soil water deficits.Crop productivity and wild plant distributions are governed by the availability of soil moisture (Axelrod, 1972; Boyer, 1982; Ciais et al., 2005). The impact of drought and soil water deficit in agriculture is estimated to be the largest abiotic determinant of yield (Boyer, 1982; Araus et al., 2002), while drought is also considered a primary cause of speciation and adaptation in nature (Stebbins, 1952). Dehydration avoidance and other drought adaptive strategies permit plants to survive or maintain growth during periodic droughts (Blum, 1996; Chaves et al., 2003; Chaves and Oliveira, 2004). Specifically, phenotypic plasticity of stomatal conductance, water foraging, and growth traits (among many others) may effectively maintain homeostasis of leaf water potential despite soil water deficits.Leaf water potential is a bellwether of the physiological impact of water deficit (Jones, 2007). Under drought, decreasing water availability results in reduced leaf water potentials and a sequence of physiological responses including reduced photosynthesis, growth rate, and ultimately, fitness (Taiz and Zeiger, 2014). Plants therefore seek to maintain homeostasis of leaf water potential, with the highest (least negative) values supporting the most efficient functioning of photosynthesis and other metabolic processes in most species (Lawlor and Fock, 1978; Turner and Begg, 1981; Kramer and Boyer, 1995; Cornic and Massacci, 1996; Jones, 2007). Plants that exhibit dehydration avoidance strategies compensate for soil water deficit through phenotypic plasticity of gene expression (Verslues et al., 2006; DesMarais and Juenger, 2010; DesMarais et al., 2013; Lovell et al., 2015) and downstream physiological phenotypes (Levitt, 1980), among others.To understand plant stress responses, it is critical to determine the physiological and genetic underpinnings of drought adaptation in both field and laboratory conditions (Travers et al., 2007; Gaudin et al., 2013). A common finding among such studies is that physiological and gene expression responses to drought vary considerably depending on the severity and temporal dynamics of drying soil (Chaves et al., 2003; Barker et al., 2005; Malmberg et al., 2005; Mittler, 2006; Mishra et al., 2012). Natural soil moisture variation, which has shaped adaptive responses to drought in wild populations, is not necessarily recapitulated by controlled (often, “shock”) laboratory experiments. For example, single abiotic stresses rarely occur in isolation in the field (Mittler, 2006). Instead, wild and crop plants respond to the combination of diverse stressors such as drought, heat, and salinity, simultaneously and at both molecular (e.g. Rizhsky et al., 2002; Rizhsky et al., 2004; Suzuki et al., 2005) and physiological (e.g. Heyne and Brunson, 1940; Craufurd and Peacock, 1993; Machado and Paulsen, 2001) levels. Therefore, inquiries into evolved plant stress responses are perhaps best served by experimental conditions that emulate selective agents in the field. Given that the extent and severity of stress causes qualitatively different physiological responses, it is not surprising that several studies have found relatively weak genetic correlations between laboratory phenotypes and those collected in the field (e.g. Weinig et al., 2002; Malmberg et al., 2005; Anderson et al., 2011; Mishra et al., 2012).Soil properties and biota can also affect plant growth and physiology (Meisner et al., 2013; Schweitzer et al., 2014), which may be exacerbated by contrasts between growth in potting mix or in native soil (Rowe et al., 2007; Heinze et al., 2016). The observation that field-grown plants have different root systems and greater total water storage than those in greenhouse pots is of particular importance to water relations (Poorter et al., 2012a). Short-term drought stress in the field may be buffered by access to larger volumes of soil and more complex root-soil-water dynamics, conditions poorly represented in most controlled settings.The field of experimental design has been fundamentally shaped by a central problem of biology: that it is notoriously difficult to control environmental factors in the field (Jones, 2013). A classic solution is to increase biological replication, but this is generally not feasible with costly and time-sensitive physiological and genetic assays (Poorter et al., 2012b; Marchand et al., 2013). Despite these difficulties, understanding the effects of drought in field conditions is necessary because it is in these settings that yield is impacted and selection is acting to shape adaptive responses to stress. Here, we determine how the interplay between drought severity, planting condition (e.g. field, potted, greenhouse) and sampling timing impacts physiological and genomic responses to drought in the C₄ perennial grass, Panicum virgatum (switchgrass). To accomplish this, we used observations collected from clonally replicated individuals of the “AP13” switchgrass genotype (derived from the Alamo cultivar), which is the genome reference for this important biofuel crop and dominant member of mesic tall grass prairie ecosystems. The Alamo cultivar is a southern lowland accession that has high vigor and performance across a variety of climatic conditions. Replicates were grown in three separate soil moisture manipulation experiments with distinct rooting environments: in medium sized pots in a greenhouse, in large containers in a field setting, and in native soil under rainout shelters. In all three of these experiments, we collected leaf-level physiological and whole-genome gene expression data from droughted and control plants.Combined, the three experiments represent contrasts in drought experimental manipulations (i.e. the extent, timing, and duration of drought), plant characteristics (i.e. age, maturity, and size), and broadly fit with the concepts of best practice for physiological analysis of drought responses (Poorter et al., 2012b). Contrasting these experimental design considerations allows us to address how edaphic and climactic conditions impact links between gene expression and physiological phenotypic plasticity. Specifically, we assessed three fundamental questions pertaining to physiological genomics in the field: (1) How consistent is phenotypic plasticity to drought across experiments? (2) Which soil moisture deficit responses vary across sites, years, and timing of sampling? (3) How does plasticity of physiological and gene expression phenotypes covary within and across experiments? To assess these questions, we tested how leaf physiology and whole-genome gene expression responded to the effects of drought treatments, leaf water potential, and sampling time (midday and predawn). These analyses permitted inference of the number, relative effect size, and identity of differentially expressed (plastic) genes. Overall, our results suggested that differences in leaf water potential and diurnal patterns were the major drivers of gene expression variation. Furthermore, we observed consistent physiological plasticity across greenhouse dry-down and field precipitation manipulation experiments, but extreme variability in the number of differentially expressed genes.     

Role of TonB1 in Pyoverdine-Mediated Signaling in Pseudomonas aeruginosa

Pyoverdines are siderophores secreted by Pseudomonas aeruginosa. Uptake of ferripyoverdine in P. aeruginosa PAO1 occurs via the FpvA receptor protein and requires the energy-transducing protein TonB1. Interaction of (ferri)pyoverdine with FpvA activates pyoverdine gene expression in a signaling process involving the cytoplasmic-membrane-spanning anti-sigma factor FpvR and the sigma factor PvdS. Here, we show that mutation of a region of FpvA that interacts with TonB1 (the TonB box) prevents this signaling process, as well as inhibiting bacterial growth in the presence of the iron-chelating compound ethylenediamine-di(o-hydroxy-phenylacetic acid). Signaling via wild-type FpvA was also eliminated in strains lacking TonB1 but was unaffected in strains lacking either (or both) of two other TonB proteins in P. aeruginosa, TonB2 and TonB3. An absence of pyoverdine-mediated signaling corresponded with proteolysis of PvdS. These data show that interactions between FpvA and TonB1 are required for (ferri)pyoverdine signal transduction, as well as for ferripyoverdine transport, consistent with a mechanistic link between the signaling and transport functions of FpvA.Pseudomonas aeruginosa is an opportunistic pathogen that is able to cause severe infections in patients with cystic fibrosis and in immunocompromised individuals, such as burn victims. Under conditions of iron limitation, P. aeruginosa secretes an iron-scavenging compound (siderophore) called pyoverdine. Ferripyoverdine is transported back into the bacteria by an outer membrane (OM) receptor protein, FpvA. The transport of ferripyoverdine via FpvA requires energy provided by a TonB complex (36, 42, 50). TonB is an energy-transducing protein that couples the energy of the cytoplasmic membrane (CM) to a variety of OM receptors required for the import of ferrisiderophores and other molecules. TonB acts in a complex with two CM-associated proteins, ExbB and ExbD, both of which are required for full TonB function (5, 37). The TonB-ExbB-ExbD complex has been identified in many gram-negative bacterial species and is thought to be a conserved mechanism for energy transduction to OM receptor proteins (31). TonB-dependent receptors contain a conserved protein motif known as the TonB box (5). Direct interaction between TonB and the TonB box has been demonstrated for several TonB-dependent receptors (8, 26, 33, 35, 47). Mutations of the TonB box, particularly mutations that are likely to affect the secondary structure, can result in a TonB-uncoupled phenotype characterized by loss of TonB-dependent functions (ferrisiderophore transport) with no loss of TonB-independent functions, such as internalization of bacteriophage (37).The P. aeruginosa PAO1 genome contains three tonB genes, tonB1 (PA5531) (36), tonB2 (PA0197) (55), and tonB3 (PA0406) (20), encoding proteins of 342, 270, and 319 amino acids (aa), respectively. The TonB1 and TonB2 amino acid sequences display 31% identity over a section of 187 aa, but otherwise, the three PAO1 TonB proteins show similarity (30 to 40% aa identity) to each other only over short (<70-aa) regions. TonB1 is considered to be the primary TonB protein involved in iron transport in P. aeruginosa. tonB1 mutants are impaired for growth in iron-limited medium and are defective for siderophore-mediated iron transport and heme utilization (36, 50, 55). Moreover, direct interaction between TonB1 and the ferripyoverdine receptor FpvA has been demonstrated in vitro (1). The tonB2 gene is not required for growth in iron-limited medium (55). However, tonB1 tonB2 double mutants grow even less well under iron limitation than tonB1 mutants, indicating that TonB2 may be able to partially complement TonB1 in its role in iron acquisition (55). The tonB3 gene is required for twitching motility and assembly of extracellular pili (20), but it is not known whether TonB3 has a role in iron acquisition. Genes encoding ExbB and ExbD proteins are located directly downstream of tonB2 (55) but are not found in association with tonB1 or tonB3.Besides its role in ferripyoverdine transport, FpvA is part of a signal transduction pathway and thus belongs to a subset of TonB-dependent receptors known as TonB-dependent transducers (reviewed in references 23 and 51). Mutational analysis has shown that the ferripyoverdine transport and signaling roles of FpvA are separate and discrete functions (21, 46). Besides FpvA, the signal transduction pathway involves a CM-spanning anti-sigma factor protein, FpvR, and (ferri)pyoverdine. (It was previously thought that both ferri- and apopyoverdine could bind FpvA (43). However, it was recently reported that only ferripyoverdine is able to form a high-affinity interaction with FpvA (13). The designation (ferri)pyoverdine will be used here to represent the active signaling molecule. FpvA and (ferri)pyoverdine regulate the activity of FpvR, which in turn regulates the activities of two extracytoplasmic function family sigma factors, PvdS and FpvI (3, 25). Upon binding of (ferri)pyoverdine to FpvA, a signal is transmitted to FpvR, resulting in activation of PvdS and FpvI. Activation of PvdS is required for maximal synthesis of pyoverdine itself, as well as two secreted proteins (25). Activation of FpvI leads to increased expression of fpvA (3, 39). In the absence of pyoverdine-mediated signaling, caused by the lack of FpvA or pyoverdine or overexpression of FpvR, suppression of PvdS- and FpvI-dependent gene expression occurs (3, 25), and this is associated with proteolysis of PvdS (49).Analogous siderophore transport and signaling systems involving an OM TonB-dependent transducer, a CM-bound anti-sigma factor, and an extracytoplasmic function family sigma factor have been described in other bacteria, including the ferric citrate (Fec) system in Escherichia coli and the pseudobactin (Pup) system in Pseudomonas putida (reviewed in reference 6). The TonB protein is required for signaling in both the Fec (14, 33) and Pup (24) systems. Similarly, a TonB system is required for hemophore transport and signaling in Serratia marcescens (4). The aim of this study was to investigate whether TonB was required for pyoverdine-mediated signaling in P. aeruginosa, and if so, to identify which of the three TonB proteins was involved.     

The demonstration of phospholipids in certain cells in neurosecretory nuclei in the rat with Baker's acid haematein after fixation in glutaraldehyde-formol-calcium

Summary With Baker's acid haematein test certain ganglion cells in the brain, their processes and, at some sites, glial cells around blood vessels stain dark blue. This article describes a study of the Baker-positive cells which occur in and around the neurosecretory nuclei. By substituting formol-calcium fixation with glutaraldehyde-formol-calcium fixation shrinkage in brain tissue is completely avoided. If such fixation is used the argument that positive staining of ganglion cells with Baker's method only indicates that these are shrunken neurons can no longer be maintained. A comparative histological study, especially of Baker's technique and controlled chromation (Elftman) showed that the Baker-positive cells contain a phospholipid, probably bound to a protein, as a labile compound, which is easily lost. We found that to immobilize and localize this labile compound in the ganglion cells the technique of fixation and the pH during chromation (which should be around 3.8) are of fundamental importance. Only under these conditions is the complex sufficiently immobilized to allow of its demonstration with acid haematein. These requirements are now completely met if Baker's acid haematein technique is used. The article stresses that only prefixed and chromated frozen sections can be used for this method, thus avoiding shrinkage and non-specific staining of proteins. The modified Baker method as used by us gives constant and reproducible staining and is described in this article. The functional significance of the Baker-positive reaction in some ganglion cells in the n. s. nuclei or glial cells around blood vessels is not dealt with in this article.     

Potassium modulation of methionine uptake in astrocytes in vitro

Methionine participates in a large variety of metabolic pathways in brain, and its transport may play an important regulatory role. The properties of methionine uptake were examined in a preparation of neonatal rat brain astrocytes. Uptake is linear for 15 minutes, up to 2.5 M. At steady state conditions, methionine is concentrated 30–50-fold. Measured methionine homoexchange accounts for a significant fraction of uptake at concentrations greater than 10 M. We recently reported that methionine uptake is decreased by elevations in extracellular K⁺. Potassium induced efflux cannot account for this apparent effect; and thus for concentrations less than 2.5M, and for short times of incubation, measured rates of methionine uptake represent unidirectional flux. At extracellular concentrations of K⁺ equal to 6.9 mM, the apparentV _max of methionine transport is 182 pmol/min/mg protein, and theK _m is 1.3 M. Where K⁺ is shifted to 11.9 mM, theK _m remains unchanged, and theV _max is reduced by half.     

Mechanics of tether formation in liposomes

It is well-known that a "tether" may be drawn out from a pressurized liposome by means of a suitably applied radial-outward force applied locally to the lipid bilayer. The tether is a narrow, uniform cylindrical tube, which joins the main vesicle in a short "transition region." A first-order energy analysis establishes the broad relationship between the force F needed to draw the tether, the radius R0 of the tether, the bending-stiffness constant B for the lipid bilayer and the membrane tension T in the pressurized liposome. The aim of the present paper is to study in detail the "transition region" between the tether and the main vesicle, by means of a careful application of the engineering theory of axisymmetric shell structures. It turns out that the well-known textbook "thin-shell" theory is inadequate for this purpose, because the tether is evidently an example of a thick-walled shell; and a novel ingredient of the present study is the introduction of elastic constitutive relations that are appropriate to the thick-shell situation. The governing equations are set up in dimensionless form, and are solved by means of a "shooting" technique, starting with a single disposable parameter at a point on the meridian in the tether, which can be adjusted until the boundary conditions at the far "equator" of the main vessel are satisfied. It turns out that the "transition region" between the tether and the main vessel is well characterized by only a few parameters, while the tether and main vessel themselves are described by very simple equations. Introduction of the thick-shell constitutive relation makes little difference to the conformation of and stress-resultants in, the main vessel; but it makes a great deal of difference in the tether itself Indeed, a kind of phase-change appears to take place in the "transition region" between these two zones of the liposome.     

Phytoferritin in plastids of the cambial zone of willow

Summary Crystalline and paracrystalline arrays of electron-opaque granules have been found in plastids of the cambial zone and its immediate derivatives in crack willow (Salix fragilis L.). These granules have a diameter of 55 to 70 Å and, when in crystalline arrangement, show a centre to centre spacing of 100 Å with adjacent, slightly curved, or linear rows running parallel. The 70 Å particles have a substructure of four to six subunits 15 Å in diameter. These units are arranged around an electron-translucent core 20 Å diameter. It is suggested that this complex is phytoferritin. It is assumed that the electron-translucent area around the opaque granules represents the proteinaceous shell characteristic of both plant and animal ferritin as described by other authors. The phytoferritin is commonly found spread in a thin, regular, array over the surface of plastoglobuli in the plastids.It is further suggested that the phytoferritin is an iron-protein complex which allows the plant to store iron in non-toxic form. This theory would be in accord with the presence of phytoferritin in plastids which appear to be morphologically mature but which, on account of their position within the stem, would not be expected to be photosynthetically very active.     

The role of migration in the genetic structure of populations in temporally and spatially varying environments II. Island Models

The Island Model introduced by Sewall Wright (1951) has proven to be a useful construction for studying the interaction of genetic drift, population subdivision, and mutation. Interest in the model has recently increased because of its relevance to certain questions involving the rate of differentiation of sub-populations under the neutral allele hypothesis (e.g., Smith, 1970; Latter, 1973). It is perhaps the only realistic population structure in which the test for neutrality proposed by Lewontin and Krakauer (1973) is valid (Lewontin and Krakauer, 1975). If data from natural populations is to be compared to the predictions of the Island Model, it is desirable to have an alternative model with the same migration pattern but with natural selection operating. In this paper one such model will be introduced where the stochastic element comes from random fluctuations in the environment rather than from genetic drift. The model is a direct extension of the one in the previous paper in this series (Gillespie, 1975) which dealt with a population which is subdivided into two patches with restricted migration between them.     

Genetics of esterases in Drosophila. IV. Slow-migrating S-esterase in Drosophila of the virilis group

A slow-migrating -esterase (S-esterase) is described which has been detected in Drosophila montana, Drosophila imeretensis, and some stocks of Drosophila virilis when mixtures of - and -naphthyl acetate are used as substrates in histochemical reactions after electrophoresis. Sexual dimorphism for S-esterase has been demonstrated. This esterase is contained in male genitalia only, predominantly in the ejaculatory bulb (waxy plug). It appears 3–4 days after emergence of flies. In hybrids between S⁺ and S⁰ species, the activity of the slow esterase is either decreased or inhibited. An autonomous synthesis of the S-esterase in the ejaculatory bulb was established by transplantation of imaginal genital discs into larvae of different Drosophila stocks. Based on analysis of physicochemical and immunochemical properties, S-esterase is suggested to be an independent fraction of esterase, possibly dimeric, which does not cross-react with -esterase antiserum.